DNA Size Selection System Operations Manual

DNA Size Selection System Operations Manual
DNA Size Selection System
Operations Manual
Software v.6.00
Cassette Definition Set 7
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Sage Science Inc.
Suite 3150
500 Cummings Center
Beverly, MA. 01915
© 2013 Sage Science, Inc. All rights reserved. Sage Science and Pippin Prep are
trademarks of Sage Science, Inc.
All other brands and name mentioned herein are property of their owners.
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TABLE OF CONTENTS
1
2
3
4
5
6
INTRODUCTION ................................................................................................... 1-1
SAFETY AND PRECAUTIONS ............................................................................. 2-1
UNPACKING AND INSTALLATION ..................................................................... 3-2
ACHIEVING BEST RESULTS FROM THE PIPPIN PREP ................................... 4-1
OPTICAL CALIBRATION ..................................................................................... 5-1
PREPARING A CASSETTE .................................................................................. 6-1
6.1
Visually Inspect the Cassette ........................................................................................................ 6-1
6.2
Prepare the Cassette for Loading ................................................................................................. 6-3
6.3
Continuity Test ................................................................................................................................ 6-4
7
8
9
SAMPLE PREPARATION ..................................................................................... 7-1
LOADING SAMPLES ............................................................................................ 8-1
RUNNING A PROTOCOL ..................................................................................... 9-1
9.1
Overview .......................................................................................................................................... 9-1
9.2
Starting a Run .................................................................................................................................. 9-1
9.3
Monitoring a Run ............................................................................................................................. 9-4
9.4
Log Files .......................................................................................................................................... 9-6
10 SAMPLE COLLECTION ..................................................................................... 10-1
10.1
Overview ...................................................................................................................................... 10-1
10.2
Intrinsic Sample Recovery on the Pippin Prep ........................................................................ 10-2
10.3
Improving Product Recovery with the Field Reversal ............................................................. 10-3
10.4
Improving Product Recovery of Larger Fragments with a Surfactant Rinse........................ 10-4
10. 5
Improving Product Yield by Selecting a Wider Size Range ................................................... 10-4
11 WRITING AND EDITING A PROTOCOL ............................................................ 11-1
11.1
Overview ...................................................................................................................................... 11-1
11.2
Programming a New Protocol – Quick Guide .......................................................................... 11-2
11.3
Selecting a Cassette Definition ................................................................................................. 11-4
11.4 Selecting a Run Time ................................................................................................................... 11-5
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11.5
Assigning a Reference Marker – external standard ................................................................ 11-6
11.6
Assigning Reference Markers – internal standards ............................................................... 11-7
11.7
Programming a “Tight” Collection ............................................................................................ 11-8
11.8
Range Mode– programming broad size range extractions .................................................... 11-9
11.9
Time Mode – programming timed cuts ................................................................................... 11-10
11.10 Peak Capture Mode – programming peak/band collections ............................................... 11-11
11.11
Warnings and Indicators ........................................................................................................ 11-12
12
13
14
15
16
SYSTEM VALIDATION ....................................................................................... 12-1
SYSTEM OPTIONS TAB .................................................................................... 13-1
RUNNING IN MANUAL MODE ........................................................................... 14-1
ANALYZING RUNS - LOG REVIEW TAB .......................................................... 15-1
MANAGING FILES -- FILE MANAGER TAB ...................................................... 16-1
16.1
Overview ...................................................................................................................................... 16-1
16.2
File Types ..................................................................................................................................... 16-2
17 UPGRADING SOFTWARE ................................................................................. 17-1
17.1
Extracting the Files to a USB flash drive ................................................................................. 17-1
17.2
Upgrading the Pippin Instrument Software ........................................................................ 17-3
18
19
20
21
WARRANTY AND SERVICE .............................................................................. 18-1
ORDERING INFORMATION ............................................................................... 19-1
INSTRUMENT SPECIFICATIONS ...................................................................... 20-1
SPECIFICATIONS FOR GEL CASSETTES ....................................................... 21-1
21.1
3% Agarose, ethidium bromide, 90 – 250 bp, Marker C ......................................................... 21-2
21.2
2% Agarose, ethidium bromide, 100 – 600 bp, Marker B ....................................................... 21-3
21.3
2% Agarose, EtBr, 25 l elution well, 50 – 600 bp, Marker S (for EpiCentre ScriptSeq v2) 21-4
21.4
1.5% Agarose, ethidium bromide, 250 bp – 1.5kb, Marker A ................................................. 21-5
21.5
0.75% Agarose, ethidium bromide, 2 – 8kb, Marker D ........................................................... 21-6
21.6
3% Agarose, Dye-Free, 90 bp-250 bp, Marker F (internal standards) ................................. 21-8
21.7
2% Agarose, Dye-Free, 100 bp-600 bp, Marker L (internal standards) ................................ 21-9
21.7
2% Agarose, Dye-Free, 100 bp-600 bp, 25 l elution vol., Marker G (int. stds) ............... 21-10
21.9
1. 5% Agarose, Dye-Free, 250 bp-1.5 kb, Marker K (internal standards) ......................... 21-11
21.8
2% Agarose, Dye-Free, 100 bp-600 bp, Marker E (external
marker) ......................... 21-12
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Introduction
Thank you for purchasing the Pippin Prep from Sage Science. The Pippin Prep is an
automated preparative gel electrophoresis system.
We urge you to read this manual to familiarize yourself with the system’s capabilities
and precautions.
System Overview
There are three components to the Pippin Prep system:

Instrument – The instrument contains an epifluorescence detector, an
electrophoresis power supply, an electrode array, and a Linux-based singleboard computer. The system computer is accessed by an external LCD
monitor, mouse, and keyboard. The electrode array is located in the top of
the sliding instrument cover and is not user-accessible.

Software – System software allows the user to enter parameters that define
the DNA fragment size ranges or DNA bands that are to be collected. The
software also logs fluorescence and electrical current data which may be
analyzed in a review screen.

Gel Cassettes – 5-lane disposable cassettes are manufactured by Sage
Science. Each cassette contains precast gel and electrophoresis buffer.
One lane is typically used for running a DNA size marker. Size markers and
sample loading solution are supplied with each cassette kit. Cassettes are
available in several gel concentrations and types to accommodate different
size ranges or applications. There are two categories of gel cassette:
o
Dye-free Cassettes – The DNA size marker has been labeled with a
fluorescent dye (TAMRA). The sample DNA is not detected by the
system. These cassettes are used for size-selecting sheared DNA.
Two types of strategies are used: 1) External standard – a DNA
marker is run in a dedicated lane to determine elution timing.
Capacity is 4 samples/cassette. 2) Internal standards – DNA markers
are added to each sample to determine elution timing. Capacity is 5
samples/cassette.
o
Cassettes with intercalating dye (ethidium bromide) – The
agarose gel and running buffer contains ethidium bromide. Ethidium
bromide is an intercalating dye which allows the system to detect
DNA above a certain threshold. These cassettes can be used for
applications in which the detection of DNA bands (PCR products or
restriction digests) are collected (“band capture” protocols).
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Safety and Precautions
Icons Used
In this manual, the following icons will be used to provide the user with information
pertinent to the use of the Pippin Prep.
Caution! Warns the user that injury or instrument damage may occur if the
contents of the warning are not properly followed.
High Voltage! Warns of the risk of electrical shock if the contents of the
warning are not properly followed.
Important! Provide important information about the proper use of the
system that may influence the quality of the result.
Information. Provides additional information regarding the function of the
system or applications for which is used.
Safe Use Guidelines
The Pippin Prep system is designed to operate under the following environmental
conditions:






Pollution Degree 2
Installation category 2
Altitude 2000m
Indoor use
Ambient temperature 17-32oC
Humidity 10-80%, non-condensing
Standard laboratory precautions should be taken when handling Pippin Prep Gel
cassettes and operating the Pippin Prep:


Wear a lab coat, safety glasses, and gloves.
Use in proximity of an eye wash station and/or running water.
Important! For ethidium bromide-containing gel cassettes, running buffer and spare
electrophoresis buffer contain 5 ug/ml of Ethidium bromide. Cassettes and buffer
should be disposed of as laboratory waste according to institutional lab policy.
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Unpacking and Installation
Unpacking the Pippin Prep
The Pippin Prep Instrument
The Pippin Prep instrumentation is shipped in two boxes: one will contain the Pippin
Prep and Accessories and the second box will contain the computer monitor in the
manufacturer’s original packaging. With the boxes in the upright position, open and
confirm that the following items are enclosed:
Monitor



LCD computer monitor
Video cable
Power cord
Pippin Prep


Pippin Prep Instrument
Accessory box
o Computer keyboard, USB
o Computer mouse, USB
o Power supply
o Plug adapter (adapts non-U.S. power cords, see Figure 3.1)
o Power cord
o Rinse cassette (for maintenance of electrodes)
o Calibration cassette (for setting optical baseline before each
run)
Figure 3.1. Adapter plug for non-US
power cord installation.
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Setting up the Pippin Prep
1. Remove the accessory box located on inside edge of box at the front end of the
instrument. Remove the keyboard, mouse, and instrument power supply from their
packaging.
2. Firmly grip both sides of the instrument and lift it from the foam packaging insert. The
Pippin Prep weighs approximately 14 lbs. Place the Pippin on a table or bench top.
3. Remove the computer monitor from the manufacturer’s box and connect the monitor
to Pippin Prep using supplied video cable. Connect monitor to power using power
supply supplied with monitor.
4. Insert USB connector from computer key board into port located in the back of the
Instrument.
5. Connect mouse to Pippin Prep via USB or PS2 connector at back of instrument.
6. Connect monitor cable into the video port located in the back or the instrument.
7. Connect monitor to power using power supply and cords supplied with monitor.
8. Connect Pippin Prep instrument to power via supplied power supply and cable. Power
connector is at rear of instrument.
9. Press power switch located on the rear of the instrument, and wait for software to
launch (approximately 30 seconds).
The Pippin Prep is ready for use. The software should automatically launch
– allow 30 seconds.
Figures 3.2 and 3.3 on the next page show the rear panel of the Pippin Prep.
Figure 3.4 on the following page shows the front panel.
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USB Ports (4)
VGA monitor port
Power Switch
Figure 3.2. Rear Panel Ports
Power Entry Port
Mouse
Key board
Monitor Cable
Power Cable
Figure 3.3. Rear Panel Connections
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Figure 3.4. Pippin Prep front panel.
USB Port
Indicator Light:
Green when electrophoresis is
underway (protocol is running).
Indicator Light:
Blue when instrument is
ready for use (power is on,
and software is active).*
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Unpacking Gel Cassettes
Gel cassettes are shipped in boxes in the following configurations. Ensure boxes are
in the upright position and confirm that following contents are present. DNA size
markers and loading solution should be stored at 4oC. (Storage of electrophoresis
buffer at 4oC is optional.) The cassettes should be stored at room temperature:

10 cassettes with reagents to run 40 samples
o
o
o
10 foil-sealed gel cassettes (store at R.T.)
1 reagent kit for 10 cassettes (store at 4 oC)

40 ml of running buffer
 440 μl DNA size markers (external marker)
 500 μl loading solution (external marker)
-or 440 μl marker/loading solution mix (internal standard)
1 package of adhesive tape for sealing elution wells
o
Important! DNA size markers and loading solution should be stored at 4 C. Loading solution
is viscous should be equilibrated at room temperature prior to use.
Important! Cassette should be stored at room temperature in the sealed foil packaging.
They are light sensitive and should not be removed until prior to use. Shelf life is one year.
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Achieving Best Results from the
Pippin Prep
The Pippin Prep extracts narrow or wide DNA size distributions more reproducibly
and with higher yield than manual preparative gel techniques. However, due to the
unique design of the pre-cast gel cassette, there are key differences between running
the Pippin and other agarose gel methods.
Successful Operation
It is recommended that users closely follow the instructions outlined in the following
sections (Section 5-8) until they become thoroughly familiar with the system. The
Quick Guide (included with cassettes) provides protocol reminders, but is not
intended for beginning users.
The keys to successful operation are:




Optical Calibration (Section 5)
Preparation of the cassettes (Section 6)
Purity of the input sample (See Section 7)
Sample loading (See Section 8)
Common Misconceptions
Users should be aware of the following characteristics of the Pippin Prep System.
These are part of normal operation of the system, but may seem counterintuitive at
first.
Narrowest is not always the best.
The Pippin Prep can produce very narrow size distributions from sheared
genomic DNA. However, narrower size distributions will necessarily mean that
a smaller fraction of the input DNA will be recovered. Users should broaden
their collection ranges if the default tight settings do not produce enough DNA
for their application. See Section 10.5 for details.
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DNA undergoing elution is smaller than DNA at the detector.
The branch point between the separation and elution channels is downstream
from the detector position. Figure 4.2 shows a cassette channel to illustrate.
During normal operation, the leading edge of the DNA fraction scheduled for
elution passes the detector before the start of elution (by up to several
minutes). This offset can give rise to the impression that sample elution is late,
even in runs that are functioning properly.
Figure 4.2. An illustration of the time and base pair difference between the detector
and branch point.
The rate of electrophoresis is faster in the separation channel than in the
elution channel. This may cause misconceptions with regard to the timing of
broad size selections. For instance, if one sample is programmed to select
from 400 – 600bp, and a second sample is programmed to select from 200 –
600 bp, the narrower range will finish eluting before the broader one, even
though both elutions complete collection at 600 bp.
Figure 4.3. An illustration comparing the relative rate of electrophoresis in the
separation and elution channels
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Elution creates a jump in the optical background of the sample lanes.
Since ethidium bromide is positively charged, it will migrate toward the (-) electrode during
electrophoresis – moving in the opposite direction from the DNA. During the initial phase of a
Pippin Prep run, the separation channel will become partially depleted of ethidium bromide.
However, when elution begins, a front of fresh ethidium bromide from the unused elution channel
will migrate up through the separation, past the detector, and create a jump in the ethidium
bromide background signal, which can be misinterpreted as a DNA signal. This is illustrated in
Figure 4.4.The ethidium bromide front passes the detector approximately 4-5 minutes after start of
elution. The ethidium bromide front may not be noticeable when DNA input is high.
Ethidium bromide fronts
due to elution
Figure 4.4. Relatively low input sheared DNA sample with broad size
distributions (300-500 bp) in lanes 2-5. Sample loading is the same
in all lanes but different DNA ranges were eluted. Brighter bands are ethidium
bromide fronts due to elution.
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Optical Calibration
The Pippin Prep optical system should be calibrated for the specific type of cassette to
be used (dye-free and ethidium bromide-containing). The instrument is supplied with a
calibration fixture that fits into the optical nest. If a lab is using only one type of
cassette, the optics should be calibrated daily (when the instrument is in use). If users
switch cassette type that is to be run (e.g. run a dye-free cassette and then a ethidium
bromide cassette or vice versa) the optics should always be re-calibrated prior to
running the new type.
Main Tab
Lane
Status
Panel
Graph
Display
Protocol
Status
Panel
Image
Display
Control
Panel
1. If switching from a ethidium bromide cassette to a dye-free cassette, rinse the
electrode array by filling the rinse cassette with water, placing the rinse cassette onot
the optical nest, and closing the lid for 20 seconds.
2. Press “CALIBRATE” on the Control Panel
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3. An “LED Calibration” window will launch. The “Calibration Status” field will contain the
message “Calibration not done”.
4. Place the Calibration fixture onto the optical nest, so that all five LED detectors are
covered. The dark side of the fixture must be down (closest to the LEDs):
Bottom
Top
Correct Position
on Optical Nest
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5. Close the lid.
6. Check that the LED target setting is ‘0.80’ .
Make sure ‘0.80’ is
entered in this field
7. Press “CALIBRATE”
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8. The LED photocurrents will automatically adjust to a factory setting, and the
“Calibration Status” field will contain the message “Calibration OK”.
Press “EXIT” to return to the Main Tab
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Preparing a Cassette
6.1
Visually Inspect the Cassette
Caution! Some cassettes contain the DNA-binding dye ethidium bromide. Use
appropriate protective equipment (disposable gloves, lab coat, and lab glasses) when
handling cassettes.
1.
Remove the cassette from foil packaging. Foil package is scored at one end to
allow tearing.
2. Inspect the levels of buffer in the buffer reservoirs. Tip the cassette to consolidate
any bubbles in the reservoirs, then hold the cassette in a horizontal position and look
at the reservoirs from the top and bottom edges. Reservoirs should be nearly full of
buffer and roughly equal in volume across the cassette. If the buffer level in any
reservoir appears less than 50% full (compared with its neighbors), the low reservoir
should be refilled prior to running. When refilling, a visual check of buffer levels is
sufficient -- a measured volume is not required.
(+) side of cassette
buffer volumes are Low
(-) side of cassette
Figure 6.1. Low buffer levels in cassette buffer chambers.
3. Inspect the gel columns. Look for obvious breakage of the agarose column in each
channel.
Important! If there is obvious breakage, do not use the lane. Remaining lanes can be used.
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4. Inspect bottom of cassette for bubbles in the detection region of the gel
columns. If a cassette has been jarred during shipping, the agarose column can
delaminate from the plastic bottom of the separation channel, forming a thin flat
bubble between the gel column and the bottom of the channel. If this happens in the
region used by the optical detector (see illustration), DNA detection will be extremely
unreliable. If a lane with such a bubble is used for the reference marker, the
markers will not be properly identified and sample collection will occur at the
wrong time or fail altogether. (Only the optical properties of the channel are
affected -- electrophoretic properties of the channel are unaffected, since the DNA
travels through the center of the gel column, above the bubble.) To find such bubbles,
turn the cassette upside-down, and view the bottom of the channels under a strong
light, while tilting the cassette back and forth (see Figure 5.2, below). Figure 5.3
shows the region of optical detection.
Important! If a bubble is observed, do not use the lane to run the reference marker or for peak
capture mode applications. It may be used for size-based fractionations (tight or bp range
modes).
Important! Flat bubbles between the top surface of the gel column and the plastic top of the
channel will NOT affect run quality or optical detection. They may be used for markers or
sample without any adverse effects.
Agarose Delamination
Do not use this lane for DNA marker
Optical Region
Figure 6.2. Bubble in optical path. Do not use the lane
for the DNA reference marker.
Figure 6.3. The region of optical detection
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Prepare the Cassette for Loading
1. Dislodge bubbles from behind the elution wells. Return the cassette to the rightside up position. Tilt the cassette, sample well side down, to release any trapped
bubbles behind the elution modules into the (+) buffer chambers. Gently tap the
cassette if necessary. Figure 6.4, below, shows location to check for bubbles.
(-)
(+)
Figure 6.4. A schematic of a Gel Cassette.
2. Place Cassette into the Pippin optical nest. The cassette should be placed into
the nest in the orientation shown in Figure 6.4, above, with the sample wells to the
left side of the nest. When inserting the cassette into the nest, keep the (-) buffer
chambers tilted down so that the bubbles in the elution reservoirs won’t be trapped
behind the elution modules.
Important! Be sure the cassette is fully seated into the bottom of the nest. Detection of DNA
within the cassette will fail if the bottom of the cassette is not properly seated against the optical
region.
3. Remove adhesive strips from cassette. Place one hand on the cassette, and hold
it firmly in the nest. Grab the white tabs of the tape and pull the strips firmly and
slowly toward the front of the Pippin Prep until they are removed.
4. Remove buffer from elution modules and replace with 40μl of fresh
electrophoresis buffer. Replenishing the buffer ensures proper electrophoretic
continuity. When refilling empty modules, place tip all the way into the module and
slowly fill from the bottom, withdrawing the pipette tip while dispensing, and taking care
not to generate any bubbles that can occlude the current path. Use spare buffer that
has been supplied with the cassette package, or pipette from the buffer chambers.
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Important! Make sure that the pipette tips used for step 4 extend all the way to the bottom of
the elution modules without sealing the elution port opening. If the tips seal the port opening, it
will be extremely difficult, or impossible, to empty and refill the elution module completely. Test
tip fit using the empty rinse cassette supplied with the instrument.
Note: The total volume of the elution module when filled to the top is 65l. The specified
starting volume of 40l only partially fills the module.
5. Seal the elution wells with the adhesive tape strips. Tape for sealing the elution
wells are supplied with cassette packaging. Long elutions will cause the elution wells
to overflow if not sealed. Place tape over the elution wells and rub firmly to fix the tape
in position. For best results, rub the tape at the boundary of the well with a smooth
hard object such as the back end or a lab marker pen.
6. Check the buffer levels in the sample wells. Sample wells should be completely
filled to the top with buffer. If any wells are under-filled, top them off with additional
buffer.
Note: The total volume of the sample well is approximately 70l.
6.3
Continuity Test
The continuity test measures the current in each separation and elution channel and
determines whether they are within the expected values for a successful run.
Important! Temperature affects electrical current readings. If cassettes have been
o
o
refrigerated, they will fail the current test until the cassette is at least 17 C (62 F). Should this
be the case, wait until the cassette temperature has equilibrated to room temperature and retest.
1. Press “Test”. With the cassette in the optical nest, close the lid and press “TEST” on
the controller on the Main Tab.
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2. Continuity Test will Automatically Run. The continuity test screen will launch. The
separation and elution channel test parameters are displayed and the electrical current
results will be listed after several seconds. If the test returns a “PASS” message,
press “RETURN” and continue to baseline calibration.
Failed Continuity Tests
1. A failed test is indicated by a “FAIL” message, and the failed channel is highlighted in
orange. Figure 6.5 shows a failed test screen (elution channel in lane 1).
2. If a Separation lane has failed (left column) continuity. Do not use that lane.
Remaining passing lanes can be used
3. If an Elution channel (right column) has failed continuity. Replace buffer in the
elution module of the failed lane and refill with 40 ul of fresh electrophoresis buffer
and retest the cassette. If the lane fails again, do not use the lane for samples.
However, the failed lane may be used to run an external reference marker.
Test parameters
Separation Channels
“FAIL” = do not use lane
Elution Channels
“FAIL” = use lane for reference
only or replace buffer and retest
Figure 6.5. The Continuity Test screen. The elution channel in lane 1 has failed
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Sample Preparation
Input Sample Characteristics
When running the Pippin Prep, characteristics of input DNA can affect separation
resolution and efficiency of product recovery. The following general guidelines should
be followed:

Ionic strength: The ionic strength of the sample should be lower than the
ionic strength of the buffer (80mM monovalent ions). High salt concentrations
can result in slower than expected DNA mobility.

Protein in the sample: DNA-binding proteins such as ligases or polymerases
can affect the mobility of fragments during separation. Proteins can also
reduce DNA recovery from the elution module by increasing the binding of
DNA to the ultrafiltration membrane at the back of the elution module. For best
results, samples should be de-proteinized prior to loading whenever possible.

Input DNA size distribution: A knowledge of the input size distribution is
obviously important to program accurate size selection settings. Pippin Prep
cassettes are calibrated using the Agilent Bioanalyzer to evaluate input and
product sizes, and so, for best results, input size distributions should be
evaluated using the Bioanalyzer. For low concentration samples, the Agilent
HS chip is very useful (see Technote 4 on the Sage website).
Preparing DNA Samples for the Pippin Prep
1.
2.
3.
4.
Bring DNA sample up to 30μl with TE.
Bring loading solution to room temperature.
For each sample, combine 30μl of DNA sample with 10μl of loading solution.
Mix samples thoroughly (vortex mixer). Briefly centrifuge to collect.
Recommended sample Load Guidelines
Maximum Load:
10g sheared genomic DNA*
4 g restriction/PCR fragment
Minimum Load:
low single nanograms
Optical Sensitivity
(ethidium bromide cassettes):
approx. 200ng sheared genomic DNA
approx. 10-15ng single bands, restriction
fragments, PCR products, or synthetic
DNA fragments
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Loading Samples
Proper sample loading is critical for best performance of Pippin Prep cassettes. For
maximum reproducibility and accuracy, the sample should travel through the central
section of the gel column, and should be bounded on all four sides by uniformly
conductive media – either gel or electrophoresis buffer. The goal of the loading
procedure is to produce this geometry in the sample loading well, as illustrated in the
bottom section of Figure 6.1. Properly prepared samples will be 40 ul in total volume
consisting of 30 ul of DNA mixed with 10 ul of Pippin Prep loading solution. The
loading solution contains concentrated Ficoll as a densifying agent (see following
chapter on Sample Preparation for details), and therefore the samples will sink and
form a high density layer beneath the electrophoresis buffer when pipetted slowly into
the sample wells. If there is insufficient conductive buffer over the sample, the
electrophoretic forces lines will curve upward as the sample exits the well (see Figure
6.1, upper section), and the sample will be drawn to the top of the cassette where it
can travel out of the gel into the gap between the gel column and the plastic top of the
channel. Sample moving in this gap will travel at a different rate than the sample
inside the gel column, and will lead to elution of undesired size fractions in the eluted
material. In such cases, the contaminating DNA will usually (but not always) be higher
in molecular weight than the selected DNA.
Figure 6.1. An illustration of the electrophoretic effect on a
DNA sample when a sample well in not completely filled.
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1. Re-check the buffer level in the sample wells. Make sure that sample wells are
completely full to the top with electrophoresis buffer. Top off with additional buffer, if
necessary. The total volume of the sample well is 70 ul.
2. Remove 40μl of buffer from the first sample well, and load 40μl of sample (or
marker) into that well. Take care not pierce the agarose with the pipette tip. There is
gel on all sides and bottom of the sample well. In addition, there is an agarose
“chimney” surrounding the top of the sample well that protrudes up through the
cassette cover (see Figure 10). When removing buffer, some users find it useful to
immerse the pipette tip just below the surface of the buffer and follow the liquid level
down with the tip as the buffer is removed. When buffer removal is completed, there
will be ~30ul of buffer left in the well. When adding sample, place tip of pipette just
below the surface of the buffer, and follow the liquid level up with the tip as the well
fills. Don’t be concerned if the sample well slightly overfills. The density of the sample
will allow it to sink before it can flow out of the well.
3. Repeat step 2 for remaining wells.
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Running a Protocol
The default screen on the Pippin Prep is a tabbed format. The Main Tab is the first
tab and the default screen.
9.1
Overview
The Main Tab is the screen with which users load protocols, run the instrument, and
monitor optical signal and electrophoresis.
Main Tab
Lane
Status
Panel
Graph
Display
Protocol
Status
Panel
Image
Display
Control
Panel
9.2
Starting a Run
1. Go to the “Main” Tab.
2. If necessary, select the protocol to be run from the “Protocol Name” drop down menu
in the Protocol Status Panel. The last protocol that has been programmed will
automatically be loaded. See Chapter 11 (Writing and Editing a Protocol) for
instructions on programming.
Note: The instrument optical array must be calibrated daily before running samples. A
calibration cassette is provided with each Pippin Prep instrument.
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3. A properly prepared cassette (Chapter 6) with loaded samples should be in the optical
nest. Typically, users will load samples while the cassette is on the nest (see Chapter
8). Ensure that adhesive tape has been placed over the elution wells.
4. Press “START” on the Control Panel.
5. An “LED Calibration” window will launch (see Chapter 5 for details).
Important! The Pippin Prep optical system should be calibrated for the specific type of cassette
to be used (dye-free and ethidium bromide-containing). The instrument is supplied with a
calibration fixture that fits into the optical nest.. If a lab is using only one type of cassette, the
optics should be calibrated daily (when the instrument is in use). If users switch cassette type
that is to be run (e.g. run a dye-free cassette and then a ethidium bromide cassette) the optics
should always be re-calibrated prior to running the new type.
6. If the instrument optics have not yet been calibrated, or if the a new cassette type is
being run:
a. remove the cassette
b. and place the calibration fixture into the optical nest
c. select the cassette type,
d. press “CALIBRATE”.
e. The LED photocurrents will automatically adjust to a factory setting, and the
“Calibration Status” field will contain the message “Calibration OK”.
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7. If the optics have already been calibrated, press “START”.
8. The LED Calibration will close.
9. On the Lane Status Panel, the “Separate” indicators will turn green (Section 9.3).
10. On the Protocol Status Panel, the progress clock will begin and the progress block will
appear (Section 9.3).
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Monitoring a Run
The Lane Status Pane has three indicator lights per lane. When the instrument is
idle, all “Idle” indicator lights are light gray.
When a protocol is running, the following indicator panel will be displayed:

Green, Separation. If a lane is separating fragments, the “Separate” indicator will be
green.

Orange, Elution. If a lane is eluting a size range, the “Elute” indicator will be
orange, and the elution timer will be active.
Note: If there is a time value in the “Elution Timer” field, and the “Separate” indicator is green,
then the elution has been completed.
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The Elution Timer records the length of time of the elution. When elution is complete,
electrophoretic path will switch back to the separation channel, and the elution timer
will show the value of the total elapsed elution time.
Run progress is monitored in the Protocol Status panel. The percent complete is
indicated by a progress bar. The “Clock” fields displays the current time and date.
The “Estimated Completion Time” displays the estimated time of protocol completion.
This value is updated and more accurate after DNA markers have been detected.
The “Remote Access” indicator is activated if the instrument is being remotely
controlled from a PC (via VNC server). Contact Sage Science for instructions on
remote control.
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Log Files
At the end of every run, the Pippin Prep will automatically save a log file (.txt) which
can be viewed on the Log Review Tab (or the PC software version of Log Review). A
screen image (.png) of the Main Tab is saved after the run has been finished. Screen
images of all continuity tests and calibration tests are also automatically saved.
All files may be accessed from the File Manager Tab. The files are saved internally
on the Pippin hard drive in the directory named /home/pippin/Pippin Prep/Logs/ in a
folder with a year-month (YYYY-MM) folder name.
Log files names have the following convention:
[software version]_[year]-[month]-[day]_[hour]-[minute]-[second]_[user input protocol
name or test type].[file extention]
An example of the four types of log files, and file structure in the File Manager Tab, is
shown below.
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Sample Collection
10.1
1.
Overview
Samples can be removed from elution modules using a standard 100-200µl pipette.
Important! Make sure that the pipette tips used for collection extend all the way to the bottom
of the elution modules without sealing the elution port opening. If the tips seal the port opening,
it will be extremely difficult, if not impossible, to completely recover the contents of the elution
module. Test tip fit using the empty rinse cassette supplied with the instrument.
2.
Samples from standard cassettes without sealed elution modules will be recovered in
40-65 ul of electrophoresis buffer, depending on the length of the elution step.
Samples from cassettes with (tape) sealed elution modules will be recovered in a
fixed volume of 40 ul.
3.
When all samples are collected, remove used cassette from instrument and dispose of
it properly.
4.
Additional DNA product can be washed from the elution modules with a nonionic
detergent. After the initial product is removed (and saved), add 40 ul of TE+0.1%
Tween 20 to the elution module. After a brief incubation (~1 min.), remove the TETween solution and combine it with the initial product. In some cases (DNA > 4kb),
DNA recovery from the Pippin Prep can be doubled by this procedure.
Tween wash is especially important for achieving a good recovery of samples that
have not been deproteinized.
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Important! Eluted samples should not be left in the cassette for longer than a couple of hours
to avoid poor recovery (due to adsorpsion or diffusion).
Important! Used cassettes should never be allowed to sit in closed instruments for long
periods of time (e.g., overnight). Under those conditions, humidity from the cassette reservoirs
can accelerate corrosion of the electrode assembly, located in the sliding cover.
Information. Electrophoresis buffer is 50 mM Tris, 30 mM TAPS, 0.1 mM EDTA. In cassettes
with Ethidium bromide, the concentration is 5 ug/ml. During elution, EDTA does not rapidly
equilibrate across the ultrafiltration membrane of the elution module, and so the final
concentration of EDTA in eluted samples is elevated to 1-2 mM
Information: Eluted samples can be used directly for ligation and amplification without buffer
exchange.
10.2
Intrinsic Sample Recovery on the Pippin Prep
Intrinsic DNA recovery in Pippin Prep cassettes is determined by running known
amounts of a plasmid restriction digest on the Pippin Prep, collecting a broad range of
fragments from the digest, and comparing the input and product profile quantitatively
using the Agilent Bioanlyzer 2100. The intrinsic recovery of DNA fragments on all
Pippin cassette types is between 50-80%.
Figure 10.1, below, shows the recovery of a 5kb band from a 0.75% cassette.
Figure 10.1. An example of the intrinsic recovery of 5 kb band on a 0.75%
agarose cassette.
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Improving Product Recovery with the Field Reversal
Providing a brief current reversal at the end of a sample elution can improve sample
recovery by up to 20%. This option is set in the System Options Tab of the Pippin
Software . The use of this option is recommended. Figure 10.2, below, shows the
improvement that can be achieved. Figure 10.3 shows the setting option in software.
The default setting is off. When activated, the current will be reversed on all
subsequent runs until the user changes the setting through the System Options tab.
Figure 10.2. Improving recovery with a 5 sec.current reversal. A 40 minute elution
of a plasmid restriction digest was run, and each data point represents
an individual band.
Press button and then press (“Accept”)
Figure 10.3. System Options Tab with expanded view of Reverse Field Option.
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Improving Product Recovery of Larger Fragments
(>500bp) with a Surfactant (Tween) Rinse
Sample recoveries of targets over 500 bp can be improved by using a surfactant
rinse:
1. Remove the sample from the elution well at the end of the run.
2. Add 40ul of Tween solution (supplied with the cassettes) to the elution well.
3. Wait 1 minute.
4. Remove solution and pool with the original extracted sample.
10. 5
Improving Product Yield by Selecting a Wider Size
Range
The Pippin Prep can produce very narrow size distributions from sheared genomic
DNA. However, narrower size distributions will necessarily mean that a smaller
fraction of the input DNA will be recovered. Users should broaden their collection
ranges if the default tight settings do not produce enough DNA for their application.
Figure 10.4 shows an example of the effect of size range selection vs. yield.
Figure 10.4. Increasing yield by widening the size selection range.
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Writing and Editing a Protocol
The default screen on the Pippin Prep is a tabbed format. The Protocol Editor is the
second tab.
11.1
Overview
The protocol editor tab is the screen with which users create new protocols, or edit
existing protocols, for DNA size selection. A protocol is created for a cassette type
(within the applicable target range) and is saved as a named protocol file with a
“.ppprot” file extension.. When running a cassette, an appropriate protocol file is
selected and applied to the run. A protocol file may be applied to any run, provided
the cassette type is correct.
Protocol
Editor Tab
Protocol
Name
Cassette
Type
Selector
Run Time
Selector
Protocol
Parameters
Warnings
Text Box
File
Commands
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Protocol Parameters Detail
The protocol parameters section is shown below. Users will select a programming
mode for each sample, and a lane to designate as a reference. Then, the size
selection values are entered either as base pair (BP) values, or time values
(hr:min:sec in “Time” mode). For “Peak” programming mode, users enter a base pair
value into the “BP Thresh” field, the instrument will collect the next peak that it detects
after that threshold has been reached.
Individual lanes may have different parameters and different modes.
Programming
Modes
11.2
Ref. Lane
Indicators
Base Pair
(BP) values
Time
values
Programming a New Protocol – Quick Guide
The following is a summary of the steps required to program a protocol. Detailed
instructions are provided in the subsequent sections.
1. Click the “Protocol Editor” tab along top of the screen.
2. Select “NEW”, to create a new protocol. To edit an existing protocol, press the
“LOAD” button to select the protocol to be edited.
3. Select a Cassette Type from “Cassette” menu, by clicking the folder icon. Protocol
fields cannot be edited unless a cassette type has been selected.
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4. Adjust the run time value, if necessary.
5. Program the size selection protocol, and assign the DNA reference marker lane.
There are four modes of programming on the Pippin Prep:

Tight – collects minimum allowable distribution range of DNA fragments using
the median target base pair value.

Range – allows users to select the range to be collected using starting and
ending base pair values.

Time – allows users to program extractions using the starting and ending elution
time (hr:min:sec) values only (a reference DNA marker is not used)

Peak – collects the next peak (restriction fragment or PCR band) after the set
threshold base pair value has been reached.
Note: Programming modes may be independently applied to individual sample lanes
6. Enter Sample ID or description (optional).
7. Press “Save As” button, enter a name for the protocol.
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Selecting a Cassette Definition
Cassettes types are selected from the “Cassette” drop down menu.
Cassette files are presented in a list. The appropriate cassette type to select is base on
the cassette product that has purchased, and in some instances the application to be
used (i.e. different electrophoresis protocols are used with the same cassette product).
Highlight the appropriate cassette definition file and press “SELECT”. See Appendix A
for summary of marker types and calibration
Note: “No Overflow Detection” is a legacy designation. This refers to cassettes that are run
with open elution modules – large collections can cause modules fill and possibly overflow if
not sealed with tape. Software limits previously prevented mis-programming, and the “No
Overflow” definitions were provided for user who used tape to seal the modules. Module
sealing tape is now provided with each cassette package and is recommended for all
protocols. “No Overflow Detection” is effectively included with each other existing cassette
type (and future cassette type) but the term will no longer be used.
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11.4 Selecting a Run Time
Protocols can be run with widely variable run times – up to 7 hours for low voltage
runs. The Run Time Selector consists of a field and a check box, with the following
default settings.
The 8 hour default value in the Run Time field ensures that a run is not unintentionally
ended early. The checked box in the “End Run when Elution is Completed” field will
automatically end a run when every lane that has a programmed elution has
completed its collection.
Users may adjust the desired run time by referring to Section 21, where estimated
run times are listed for each cassette definition. Users should be aware of the
following when adjusting the run time parameters:

If a run time is entered based on run time estimates from Section 21, make
sure adequate time is added to the estimate as a safety

The “Run Time” field will take precedence—When the run time is achieved,
the run will end regardless of whether the elutions are complete.
To end a run manually, go to the Main Tab and press “STOP”. Completed elutions
may be verified in the Lane Status Panel if there is a value is in the “Elution Timer”
field and the “Separate” button is lit:
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Assigning a Reference Marker – external standard
1. Determine the lane into which the reference marker will be loaded.
2.
In the protocol editor, enter the reference lane designation into “Ref Lane” field for
every cassette lane. Click “APPLY REFERENCE TO ALL LANES”. Figure 10.1
shows the proper settings for running a DNA marker in lane 1, with programmed
“Tight” extractions in lanes 2-5.
Figure 11.1. The correct configuration for a protocol with the DNA marker in
lane 1. The values may be entered with a single step.
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Assigning Reference Markers – internal standards
1. Markers must be loaded into every lane with the sample (30 ul of sample with 10 ul of
internal standard loading solution).
2.
In the protocol editor, press “USE INTERNAL STANDARDS”. This will auto-fill the
“Ref Lane” fields with reference marker internal to the sample. The software will
determine the timing of elution based on the migration of the DNA marker within its
own lane.
3. Figure 11.2 shows the correct assignation of marker lanes for internal standard runs.
Figure 11.2. The correct configuration for a protocol using internal standards.
Important! When using internal standards all samples must be prepared the Pippin Prep
internal standard loading solution which is formulated for the cassette type and size range to
be used. The internal standard loading solution contains the markers.
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Programming a “Tight” Collection
1. Assign a reference lane. A DNA reference marker is required for tight cuts. Enter
the reference lane number into the “Ref Lane” field for the sample lane.
2. Select the “Tight” programming mode. Click the “Tight” button for the lane in which
the tight cut will be extracted.
3. Enter a value in for the “BP Target”. This value should represent the median value
for the desired size range to be collected. The actual range to be collected will autofill the “BP Start” and “BP End” fields.
4. (Optional) Enter a BP pause value. If a value is entered into the pause field, the
system will pause when it has estimated that that base pair value has been collected
during the elution of that lane. A pause will allow a user to remove two consecutive
cuts from a sample. The two cuts will usually have overlapping size distributions.
User may wish to use the pause feature for two reasons: 1) to retrieve nearly identical
cuts from the same sample, or 2) to avoid overflow of the elution module during
collections that require a long elution period. The BP pause value must be within the
extraction range (between BP Start and BP End). The pause function temporarily
turns off power to the electrodes and suspends run timers, allowing users to remove
the sample, rinse the elution well (if desired), and then resume the instrument run to
collect additional sample.
Important! A pause will require the user to manually resume the protocol from the Main
Screen controller. The instrument lid may be opened during the pause, and sample
retrieved.
5.
2
Enter Sample ID information.
1
3
4 (optional)
5
Important! Minimum size ranges are may not be the ideal for some applications. Very
narrow size ranges collect less DNA than larger ranges. Users should consider the
requirement for distribution vs. yield (see sec. 7-4).
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8. Save. After programming a new protocol, or editing an existing one, the protocol file
must be saved prior to use. If the protocol is new, press “Save”. If a protocol has
been edited, a yellow alert will be displayed in the “Protocol Changes Not Applied”
field. “Save” will save the file under the previously saved name, and “Save As” will
allow a new name for the file to be applied.
All protocol files are saved in a directory; /home/pippin/Pippin Prep/Protocols, and may
be accessed in the File Manager tab.
11.8 Range Mode– programming broad size range
extractions
1. Assign a reference lane. A DNA reference marker is required for broad range cuts.
Enter the reference lane number into the “Ref Lane” field for the sample lane.
2. Select the “Range” programming mode. Click the “Range” button for the lane from
which the cut will be extracted.
3. Enter a range values in the “BP Start” and “BP End” fields. The median target
value of the collection range will auto-fill the “BP Target” field.
4. (Optional) Enter a BP pause value. If a value is entered into the pause field, the
system will pause when it has estimated that that base pair value has been collected
during the elution of that lane. A pause will allow a user to remove two consecutive
cuts from a sample. The two cuts will usually have overlapping size distributions.
User may wish to use the pause feature for two reasons: 1) to retrieve nearly identical
cuts from the same sample, or 2) to avoid overflow of the elution module during
collections that require a long elution period. The BP pause value must be within the
extraction range (between BP Start and BP End). The pause function temporarily
turns off power to the electrodes and suspends run timers, allowing users to remove
the sample, rinse the elution well (if desired), and then resume the instrument run to
collect additional sample.
Important! A pause will require the user to manually resume the protocol from the Main
Screen controller. The instrument lid may be opened during the pause, and sample
retrieved.
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5. Enter Sample ID information.
2
1
3
4 (optional)
5
6. Save. After programming a new protocol, or editing an existing one, the protocol file
must be saved prior to use. If the protocol is new, press “Save”. If a protocol has
been edited, a yellow alert will be displayed in the “Protocol Changes Not Applied”
field. “Save” will save the file under the previously saved name, and “Save As” will
allow a new name for the file to be applied.
All protocol files are saved in a directory; /home/pippin/Pippin Prep/Protocols, and may be
accessed in the File Manager tab.
11.9
Time Mode – programming timed cuts
Important! The time mode does not reference a standard. Cassette to cassette variablity and
environmental running conditions will cause a wide variation in selection accuracy.
1. Select the “Time” programming mode. Click the “Time” button for the lane from
which the cut will be extracted. A DNA marker reference standard is not required.
2. Enter a time values in the “T Start” and “T End” fields. Sample elutions will occur
at exactly those times after the start of the run
3. (Optional) Enter a Time pause value. If a value is entered into the pause field, the
system will pause at that time after the start of a run. A pause will allow a user to
remove two consecutive cuts from a sample. The two cuts will usually have
overlapping size distributions. User may wish to use the pause feature for two
reasons: 1) to retrieve nearly identical cuts from the same sample, or 2) to avoid
overflow of the elution module during collections that require a long elution period.
The T pause value must be within the extraction range (between T Start and T End).
The pause function temporarily turns off power to the electrodes and suspends run
timers, allowing users to remove the sample, rinse the elution well (if desired), and
then resume the instrument run to collect additional sample.
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Important! A pause will require the user to manually resume the protocol from the Main
Screen controller. The instrument lid may be opened during the pause, and sample retrieved.
4.
Enter Sample ID information.
1
2
3
4
6. Save. After programming a new protocol, or editing an existing one, the protocol file
must be saved prior to use. If the protocol is new, press “Save”. If a protocol has
been edited, a yellow alert will be displayed in the “Protocol Changes Not Applied”
field. “Save” will save the file under the previously saved name, and “Save As” will
allow a new name for the file to be applied.
All protocol files are saved in a directory; /home/pippin/Pippin Prep/Protocols, and
may be accessed in the File Manager tab.
11.10 Peak Capture Mode – programming peak/band collections
1. Select a reference lane. Click the “Ref” button for the lane into which the DNA
reference marker will be loaded. A DNA marker reference is required for peak
collections.
2. Select the “Peak” programming mode. Click the “Peak” button for the lane from
which the fragment peak will be extracted.
3. Enter a value in for the “BP Threshold”. Enter a base pair value that precedes the
beginning of peak/band of the fragment to be collected. If possible, use a threshold
value that is <90% of the beginning of the leading edge of the target band. The Pippin
Prep will automatically collect the next peak that is detected. The mass of minimum
detectable band is approximately 50 ng. Peak collection cannot be used with dyefree cassettes.
Important! It is important to verify the size of the band by electrophoresis (Agilent
Bioanalyzer, agarose gel). Also, higher input mass will cause bands to migrate faster due to a
lower dye/DNA ratio. For very high loads (>0.5ug/band), choose lower threshold values.
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4. Enter Sample ID information.
2
1
3
4
5. Save. After programming a new protocol, or editing an existing one, the protocol file
must be saved prior to use. If the protocol is new, press “Save”. If a protocol has
been edited, a yellow alert will be displayed in the “Protocol Changes Not Applied”
field. “Save” will save the file under the previously saved name, and “Save As” will
allow a new name for the file to be applied.
All protocol files are saved in a directory; /home/pippin/Pippin Prep/Protocols, and
may be accessed in the File Manager tab.
11.11
Warnings and Indicators
BP Range Flag
When a Minimum Size Range has been programmed into a sample lane, the “BP
Range Flag” field for that sample will display “tight” and a green color.
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When a broad size cut range (larger than the minimum) has been programmed into a
sample lane, the “BP Range Flag” field for that sample will display “broad” and an
orange color.
Pause Enabled Indicator
If the pause feature has been entered for a sample lane, “Pause On” indicator will be
displayed.
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Warnings Text Box
If there are errors or exceptions within a programmed protocol, a message will appear
in the Warnings text box identifying the nature of the discrepancy.
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System Validation
Control DNA can be purchased from Sage Science to check the performance of the
Pippin Prep system. The Control DNA is useful to test, refine, and troubleshoot Pippin
Prep size fractionation protocols. For comparison to factory tested cassettes, an
extraction protocol a typical Agilent Bioanalyzer result is provided with the kits. Figure
12.1 shows the DNA profile and extraction protocol for the 2% agarose control kit, and
Figure 12.2 shows a typical result from a 150 bp tight extraction from the DNA control
Kit.
Pippin Prep cassettes and instruments are functionally tested using restriction digests of
genomic DNA from E. coli. For each cassette type, a different restriction digest is used,
chosen so that size distribution of the digested DNA closely matches the useful
fractionation range of the cassette, without any significant peaks or discontinuities.
Following restriction digestion, the control DNA is purified by phenol:chloroform
extraction, dialyzed, and diluted into Pippin Prep electrophoresis buffer (without Ethidium
bromide). The DNA is premixed with Pippin Prep loading solution and is provided ready
for loading – no additional loading solution should be added. The DNA concentration is 5
micrograms per 40 microliters. 40 microliters of control DNA should be used per lane.
Each tube contains sufficient volume for 16 sample loads.
Figure 12.1 Control DNA profile (with reference marker) and DNA extraction
protocol for a 2% agarose DNA control kit run.
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Figure 12.2 Example of a Bioanalyzer result for the 150 bp target of a Control
DNA run on a 2.0% Agarose Cassette
Important! Data are not intended to imply guaranteed results or performance.
Control
standards are intended to demonstrate that the Pippin Prep system is functioning as expected,
and that proper operational technique is being used.
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System Options Tab
The default screen on the Pippin Prep is a tabbed format. The Protocol Editor is the fifth
tab.
Overview
The System Options Tab allows users to set or change global configurations for the
software and Pippin Prep.
System
Options
Tab
Instrument
Name
UDP Port
Reversible
Field
Date and
Time
This screen allows users to set the following:

Instrument name: A name can be given to (or changed for) the instrument that
will be displayed in the Main Tab and in the log files.

UDP Server Port: Provides and address for remote communications between
the Pippin Prep and wireless control. Contact Sage Science for support.

Reversible Field for Sample Recovery: When activated, all sample
collections will terminate with a 5 second field reversal to improve recovery.
When deactivated, all sample collections will not include the field reversal. The
default setting is ON. When activated, the current will be reversed on all
subsequent runs until the user changes the setting through the System Options
tab.

Date/Time: This allows users to set or change the date and time settings for
the instrument. The date and time settings are recorded in log files and log file
names.
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To Change the settings, press the “System Options” Tab.
Instrument name
1. Enter a text into the “Instrument Name” field.
2. The “Settings Changed” indicator light will activate. Press “Accept” to accept the new
setting.
Reverse field option
1. Press the “Use Reverse Field for Sample Recovery” button. The indicator display will
change from dark gray to white, and display change from “OFF” to “ON”.
2. The “Settings Changed” indicator light will activate. Press “Accept” to accept the new
setting.
Important! The “reverse field” setting will apply to all runs until it is changed in this tab.
Date and time
1. Edit the date or time display directly in the “Time/Dislpay” field, or select the calendar icon
next to the field and edit the time or select a date.
2. The “Settings Changed” indicator light will activate. Press “Accept” to accept the new
setting
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Running in Manual Mode
The Pippin Prep may be run without using a programmed protocol and with manual
control.
1. Select the Manual Mode from the “Protocol Name” drop down menu in the Run File
Manager.
2. Press “Start” on the controller.
3. Sample lanes may be run individually by pressing the “Separate” button, and elutions
are carried out by pressing the “Elute” button. After elution, press the “Separate” button,
to end the elution (and return to separate mode), or press the “Idle” button to stop the
run on that lane.
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4. Press “Pause” / “Resume” and “Stop” buttons are also operative as in automated runs.
Note:. There is a delay between the time that a fragment is detected (and visible on the graph or
image) and when it is in position to be eluted. See cassette specifications at the end of this
manual to determine the best timing for manual runs.
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Analyzing Runs - Log Review Tab
The default screen on the Pippin Prep is a tabbed format. The Log Review is the third
tab.
Overview
The Log review screen is used to review data and programming parameters from
previous size selection runs. All programming parameters are displayed as originally
entered in the Protocol Editor. Optical and electrical current data from the run are
displayed in graphical windows. Specific regions of either window can be selected and
expanded as needed for detailed analyses. These data are useful for refining size
selection performance and verifying instrument performance.
Log
Review
Tab
Log file
Directory
Protocol
Name
Cassette
Type
Protocol
Parameters
DNA
Marker
Times and
Values
Optical
Signal
Current
Trace
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Opening a Log file in the Review Screen
Note: A stand-alone Review Log Program is available for PCs. Contact sage science
support.
1. Click the “Log Review” tab along top of the screen.
2. To select a log file, press the folder icon in next to the “Log File” field.
3. Select a log (.txt) file.
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4. This will populate the Review Log Screen with data from the selected run log:
Protocol
Information
DNA Marker
Times and
Values
Optical
Signal
Traces
Electrophoretic
Current Traces
(current drops
during elution)
Visualizing Pippin Run Data
The current and optical signal traces are color-coded and a key is displayed to the right
of the “Current” graph. A check mark next to each lane will toggle the visualization of
that lane on and off. The type and color of the lines may also be adjusted by clicking
inside the line field and holding the left-click on the mouse. The same styles are applied
to both the signal and current traces.
Turn line
visualization
on/off
Hold left-click on
mouse to change
line color or style
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Signal Graph
The “Signal” graph allows users to closely inspect the elution results of a Pippin run from
a run log. The colored horizontal traces display the actual optical signal results in mA.
The vertical red lines indicate called reference DNA marker peaks that are listed in the
DNA marker times and values list. There is an “Analyze Peaks” button to recall the
markers.
The yellow vertical line indicates the estimated base pair position at a given run time.
The indicator may be moved (see below) along the x-axis showing the relative base pair
position. If the perspective of the graph is changed (see below), the indicator will
automatically default to the center of the x-axis.
There are four icons in the upper right hand corner of the graph. The functions are listed
below.
Revert to default
view
Move yellow
base pair
indicator
Grab and move
screen
Zoom
(hold leftclick for
Options)
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Current Graph
The “Current” graph allows users to closely inspect the electrophoretic current profile of
a Pippin run from a run log. The colored horizontal traces display the actual current
signal results in mA.
The timing of an elution (switch from separation to elution) is accompanied by a 0.5 mA
drop in current.
There are three icons in the upper right hand corner of the graph. The functions are
listed below.
Revert to default
view
Saving a Review Image
Zoom
(hold leftclick for
Options)
Grab and move
screen
An image from the review screen may be captured and automatically saved by pressing
the “Snapshot” button. The .png file will date and time stamped and saved in the Pippin
log file directory.
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Managing Files -- File Manager Tab
The default screen on the Pippin Prep is a tabbed format. The File Manager Tab is
the fourth tab.
16.1
Overview
The File Manager Tab allows users to access, copy, and delete Pippin Prep files.
There are three types of files used by the system; log (text and image), protocol and
cassette type.
The screen is divided into two sub-screens; the Pippin file directory on the left, and a
target directory (portable “flash” media, i.e. USB key) on the right.
File Manager
Tab
Pippin
File
Directory
Destination
Drive File
Directory
Pippin
Files
Destination
Drive Files
File Types
File Commands
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File Types
There are three types of files that are stored in different directories:
Log Files
The log file directory is accessed by pressing the “GO TO LOGS” button.
Every run on the Pippin Prep automatically saves the following files, which are saved
with the following convention:
[software version]_[year]-[month]-[day]_[hour]-[minute]-[second]_[user input protocol
name or test type].[file extention]

Log File. This is a text (.txt) file that contains all of the data that was collected
during a run. This file can be used as a diagnostic tool with Sage Science
support personnel. Data from the log file can be viewed in the Log Review
Screen, using the PC Pippin Review application, or copied into a spreadsheet
file to recreate the data displayed in the Pippin Prep graph images.

Screen Image File. An image file (.png) of the Main Tab screen at the
completion of the run is saved. The log and screen image files

Continuity Test. After every test, an image file (.png) is automatically saved.

Calibration Test. After every test, an image file (.png) is automatically saved.
In addition, a screenshot may be manually saved as at any time during a run (as a
.png file) by pressing “Snapshot” in the Main Tab on the controller. The same file
convention is used, but with the word “Screenshot” in the file name.
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Protocol Files
The protocol file directory is accessed by pressing the “GO TO PROTOCOLS” button.
Protocol files have a .ppprot extension and are stored in a Protocols directory with the
name saved by the user. Protocols may be copied to a flash drive for archiving or
transfer to another Pippin Prep.
Cassette Files
The log file directory is accessed by pressing the “GO TO CASSETTE” button.
Cassette files have a .ppcass extension and contain the calibration parameters for the
cassettes that are indicated in the file names. Cassette files may be copied to a flash
drive and transferred to another Pippin Prep. Cassette files are not editable.
Note: Cassette calibration files may be updated during software upgrades. Users should
check release notes and save legacy cassette files if a methods have been developed with
their use.
Managing Files
The commands for managing files are found at the bottom of the File Manager Tab.
The functions are outlined below.
Creates new
folder on
attached USB
Flash drive
Copies
highlighted
file(s) from
Pippin to
flash drive
Copies
highlighted
file(s) from
flash drive
to Pippin
Deletes
highlighted
files
Safely
unmounts
flash drive
prior to
removal
Warning! Be sure to “Unmount Flash Drive” prior to removal. Failure to do so may
cause data loss. Failure to do so may cause data loss and problems with future upgrade
procedures.
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Upgrading Software
Pippin upgrade software is available for download at
www.sagescience.com/support/. The files are provided in a zip file, and the
contents must be extracted to the root directory of a USB flash drive.
17.1
Extracting the Files to a USB flash drive
1. Download the Software Upgrade package by pressing the appropriate link on
www.sagescience.com/support/ (in the Downloads section, under “Software”).
2. Press “Save File” when prompted. The zip file will likely save to a “Downloads” folder
on your computer. The file will be named pippinupgrade[version number].zip.
3. Copy the zip file to a USB drive. Make sure the file is transferred to the root directory
and not placed within a folder.
4. Right-click on the zip file, and select “Extract All....”:
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5. A window which will launch to prompt you to select a destination where to extract
files. The prompt will suggestcreating folder based on the zip file name. Delete the
destination folder name, so that only the root directory appears (E:\ in the example
below).
Correct:
Not Correct:
6. The final USB file structure should be as shown. The zip file may be deleted.
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Upgrading the Pippin Instrument Software
1. Insert the USB drive with the upgrade folder into the USB port on the front panel of
Pippin instrument.
2. From the Main Tab, press “INFO”. This will launch the information window.
3. From the Information Window, press “SOFTWARE UPGRADE”
4. Press “OK” at the warning prompt, the Software Upgrade Window will launch.
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5. Press “OK” at the warning prompt, the Software Upgrade Window will launch.
6. Press “INSTALL UPGRADE”, this will take about 5 seconds
7. Press “UNMOUNT FLASH DRIVE”
Warning! Be sure to “Unmount Flash Drive” prior to removal. Failure to do so may
cause data loss and problems with the upgrade procedure.
8. Press “EXIT” to return to Main Menu. This will install the software. Installation may
take up to 30 seconds.
9. To check that the software insturment has been update, Press “INFO” again. In the
Information window, the software version number is listed on the left side.
Note: The system should be ready to use; re-boot is not necessary.
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Warranty and Service
Warranty
Sage Science will offer a no charge repair or replacement of defective product if notified
within the warranty period and product has not been misused, altered or damaged by
disaster. This warranty is not transferable from the original purchaser to a subsequent
purchaser.
Instruments – The Pippin Prep instrument and monitor have a standard 2-year parts
and labor warranty. Instruments are subject to depot repair or replacement. On-site
maintenance or repair is not implied. Do not attempt to repair or disassemble the Pippin
Prep instrument. This will void the remaining warranty and possible create a dangerous
condition. Sage Science support staff may ask for a log file or image file to evaluate or
troubleshoot instrument issues.
High Voltage! The Pippin Prep contains high voltage apparatus. The instrument should be
repaired by authorized personnel only. Do not disassemble under any circumstance.
Cassettes – Cassettes have a 1-year warranty. If a cassette or sample lane is defective
Sage Science will replace the cassette at no cost. The defective cassette should be
disposed of as per regulations at the user’s facility. Sage Science support staff may ask
for a log file or image file to evaluate or troubleshoot cassette issues.
Maintenance
It is possible that salts will accumulate on the electrodes that are housed in the Pippin
Prep lid assembly. This may alter the performance of the instrument over time. It is
recommended that the electrodes undergo periodic rinsing. To rinse the electrodes, use
the rinse cassette provided in the instrument shipment, fill with deionized water, load on
the sample tray and close the Pippin Prep lid. A quick rinse for several second is
sufficient.
If a rinse cassette is lost or damaged contact Sage Science support for a replacement.
No other maintenance activity is required.
Cassette Disposal
Running buffer should be disposed of according to internal lab safety policy. Running
buffer contains up to 5 ug/ml of Ethidium bromide. Cassettes should be disposed of
as laboratory waste.
Note: Dye-free cassettes TAMRA dye bound to the reference marker.
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Ordering Information
Products
Catalogue No.
Instrument
BLU0001
Pippin Prep (Includes monitor, keypad, mouse and accessories)
Gel Cassettes with Ethidium Bromide– with Enternal Marker (10/pk)
Internal
Standards
Catalogue
No.
3% agarose, with EtBr,
90 – 250 bp
C
CSD3010
2% agarose, with EtBr ,
100 – 600 bp
B
CSD2010
S
SSQ2010
A
CSD1510
D
CSD7510
50 – 600 bp, 25 ml elution volume*
2% agarose, with EtBr ,
250 – 1500 bp
1.5% agarose, with EtBr ,
2 – 8 kb
07.5% agarose, with EtBr ,
Dye-Free Gel Cassettes – with Internal Standards (10/pk)
External
Marker
Catalogue
No.
3% agarose, dye-free,
90 – 250 bp
H
CDF3010
2% agarose, dye-free,
100 – 600 bp
L
CDF2010
2% agarose, dye-free,
100 – 600 bp, 25 l elution well
G
SDF2010
E
CDF1510
1. 5% agarose, low range,
250 bp – 1.5 kb
Dye-Free Gel Cassettes – with External Marker (10/pk)
2% agarose,
100 – 600 bp
External
Marker
K
Catalogue No.
CEF2010
* These cassettes were designed for use with EpiCentre’s Script-Seq kits. The elution volume in other
cassettes are 40l.
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Ordering Information Cont’
Control DNA for Performance Validation
Catalogue
No.
Control DNA for 3% Gel Cassette, (Int. Std) 90 – 250 bp, (5 g/load), 20 loads
CIS3004
Control DNA for 3% Gel Cassette, (Ext. Mrk.) 90 – 250 bp, (5 g/load), 16 loads
CON3004
Control DNA, 2% Gel Cassette, (Int. Std). 100 – 600 bp, (5 g/load), 20 loads
CIS2004
Control DNA for 2% Gel Cassette, (Ext. Mkr.)100 – 600 bp, (5 g/load), 16 loads
CON2004
Control DNA for 1.5% Gel Cassette, (Int. Std) 250 – 1500 bp, (2 g/load), 20 loads
CIS1504
Control DNA for 1.5% Gel Cassette, ( Ext. Mkr.) 250 – 1500 bp, (2 g/load), 16 loads
CON1504
Control DNA for 0.75% Gel Cassette, (Ext. Mkr.),
1 -- 10 kb, (5 g/load), 16 loads
CON7504
Control DNA for 1. 5% Band Capture, (Ext. Mkr.), 1 kb fragment, (5 g/load), 4 loads)
CBC1501
Control DNA for 0.75% Band Capture, (Ext. Mkr.), 3 kb fragment, (5 g/load), 4 loads)
CBC7501
Reagent Replacement Kits – Ethidium Bromide with External Marker
Catalogue
No.
CSD3010 reagent replacement kit for 10 cassettes, Marker C
RPK3010
CSD2010 reagent replacement kit for 10 cassettes, Marker B
RPK2010
SSQ2010 reagent replacement kit for 10 cassettes, Marker S
RSQ2010
CSD1510 reagent replacement kit for 10 cassettes, Marker A
RPK1510
CSD7510 reagent replacement kit for 10 cassettes, Marker D
RPK7510
Reagent Replacement Kits – Dye Free with Internal Standards
Catalogue
No.
CDF3010 reagent replacement kit for 10 cassettes, Marker H
RIK3010
CDF2010 reagent replacement kit for 10 cassettes, Marker L
RIK2010
SDF2010 reagent replacement kit for 10 cassettes, Marker G
RIS2010
CDF1510 reagent replacement kit for 10 cassettes, Marker K
RIK1510
Reagent Replacement Kits – Dye Free with External Marker
Catalogue
No.
CEF2010 reagent replacement kit for 10 cassettes, Marker E
REK2010
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Instrument Specifications
Specifications for Pippin Prep Instrument
Electrophoresis Voltage
100 V or 150V, constant
Typical current per sample lane
2.0 mA
Optical detection
535 nm excitation, 640 nm emission
Power Requirements
100-240 VAC, 2.5 A, 50-60 Hz
Weight
15 lbs / 7 kg
Dimensions
7H X 11W X 21D (in.) / 18H X 28W X 53D (cm)
Approvals
CE, CSA
Country of Origin
United States
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21 Specifications for Gel Cassettes
Dye-Free Cassette Specifications
Minimum Sample Load
Maximum Sample Load
2
Accuracy
Reproducibility
low single nanograms
1
2 g sheared genomic DNA
specified on following pages
3
specified on following pages
4
Sample Recovery
50-80%
1. Up to 10 ug of DNA may be loaded, however accuracy may be affected by load amounts over 2 ug and the
distribution profile of DNA fragments. See www.sagescience.com/support for instructions on using higher
amounts of DNA.
2. Deviation of actual target value from software input value divided by the actual value.
3. 2X standard deviation of replicate samples.
4. Recovery is measured using known amounts of plasmid marker ladder.
Important! DNA input samples should be purified to eliminate DNA binding proteins such as ligases,
polymerases, restriction enzymes. Bound proteins can alter mobility and reduce recovery.
Specifications subject to change without notice.
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3% Agarose, ethidium bromide, 90 – 250 bp, Marker C
21.1
Product Number:
Use Cassette Definition:
CSD3010
3% Agarose, Marker C
DNA Marker C - Typical times to detector
Estimated Collection Times for Minimum Size Range (Tight) Targets
Target
(bp)
100
Time to Time to Collect*
(min)
67
150
82
250
118
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications
Minimum Size Distribution as CV
8%
Accuracy
<5%
Reproducibility
<5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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2% Agarose, ethidium bromide, 100 – 600 bp, Marker B
21.2
Product Number:
Use Cassette Definition:
CSD2010
2% Agarose, Marker B
DNA Marker B - Typical times to detector
Estimated Collection Times for Minimum Size Range (Tight) Targets
Target
(bp)
150
Time to Time to Collect*
(min)
57
300
73
600
111
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications
Minimum Size Distribution as CV
8%
Accuracy
<5%
Reproducibility
<5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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2% Agarose, ethidium bromide, 25 l elution well, 50 – 600 bp,
Marker S (for EpiCentre ScriptSeq v2 protocol)
21.3
Product Number:
Use Cassette Definition:
SSQ2010
ScriptSeq V2
DNA Marker S- Typical times to detector
Estimated Collection Times for Minimum Size Range (Tight) Targets
ScriptSeq Step
(bp)
Pre-purification (50-500bp)
Time to Time to Collect*
(min)
125
Library purification (150-500bp)
114
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications
Minimum Size Distribution as CV
8%
Accuracy
<5%
Reproducibility
<5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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1.5% Agarose, ethidium bromide, 250 bp – 1.5kb, Marker A
21.4
Product Number:
Use Cassette Definition:
CSD1510
1.5% Agarose, Marker A
DNA Marker A - Typical times to detector
Estimated Collection Times for Minimum Size Range (Tight) Targets
Target
(bp)
300
Time to Time to Collect*
(min)
53
600
64
1,200
105
1,500
130
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications
Minimum Size Distribution as CV
8%
Accuracy
<5%
Reproducibility
<5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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0.75% Agarose, ethidium bromide, 2 – 8kb, Marker D
21.5
Product Number:
Use Cassette Definition:
CSD7510
0.75% Agarose, Marker D
DNA Marker D - Typical times to detector
Estimated Collection Times for Minimum Size Range (Tight) Targets
Target
(bp)
2000
Mean
62
Time to Time to Collect (min)
stdev
2
4000
75
3
6000
87
3
8000
93
4
Important! Input sample loading of 0.75% gel cassettes effects accuracy
In the 0.75% gel cassettes, the input load has a large effect on size selection accuracy. Lower
input loads will size select a larger target than programmed, while higher input loads will produce
a smaller target than programmed. Users should refer to the chart on the next page to determine
how much of an offset should be expected, and some method development will likely be required
to achieve desired results. Input amounts that are below 1g have not fully characterized and
may require method development by users.
Performance Specifications
Minimum Size Distribution as CV
20%
Accuracy
Dependent on input load. See chart on
following page
Reproducibility
<10%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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To use this chart: Find the desired target size on the y-axis, and follow the value to the approximate
input amount. Enter the corresponding x-axis value into the protocol editor software. Each point is an
average of four experiments. Error bars for the 5 g load represents two standard deviations.
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3% Agarose, Dye-Free, 90 bp-250 bp, Marker F (internal
standards)
Use Product Number:
CDF3010
Use Cassette Definition:
3% DF, Marker F
DNA Marker F Typical times to detector
Estimated Collection Times for
Minimum Size Range (Tight) Targets
Target
(bp)
90
Time to Time to Collect*
(min)
63
140
70
180
80
240
90
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications*
Minimum Size Distribution as CV
Sample Recovery
90-250 bp
<8%
50 – 80%
Accuracy
<5%
Reproducibility
< 5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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2% Agarose, Dye-Free, 100 bp-600 bp, Marker L (internal
standards)
Use Product Number:
CDF2010
Use Cassette Definition:
2% DF, Marker L
DNA Marker L Typical times to detector
Estimated Collection Times for
Minimum Size Range (Tight) Targets
Target
(bp)
100
Time to Time to Collect*
(min)
49
200
57
300
63
400
71
500
79
600
86
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications*
Minimum Size Distribution as CV
Sample Recovery
90-250 bp
<8%
50 – 80%
Accuracy
<5%
Reproducibility
< 5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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2% Agarose, Dye-Free, 100 bp-600 bp, 25 l elution volume, Marker
G (internal standards)
Use Product Number:
SDF2010
Use Cassette Definition:
2% DF, Marker G, small volume elution
DNA Marker G Typical times to detector
Estimated Collection Times for
Minimum Size Range (Tight) Targets
Target
(bp)
100
Time to Time to Collect*
(min)
49
200
57
300
63
400
71
500
79
600
86
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications*
Minimum Size Distribution as CV
Sample Recovery
100-600 bp
<8%
50 – 80%
Accuracy
<5%
Reproducibility
< 5%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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1. 5% Agarose, Dye-Free, 250 bp-1.5 kb, Marker K (internal
standards)
Use Product Number:
CDF1510
Use Cassette Definition:
1.5% DF, Marker K
DNA Marker K Typical times to detector
Estimated Collection Times for
Minimum Size Range (Tight) Targets
Target
(bp)
250
Time to Time to Collect*
(min)
29
300
31
600
36
1200
47
1500
50
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications*
Minimum Size Distribution as CV
250-600 bp
800-1000 bp
1200-1500 bp
<8%
<8%
<10%
50 – 80%
50 – 80%
50 – 80%
Accuracy
<5%
<7%
<10%
Reproducibility
< 5%
< 8%
< 15%
Sample Recovery
Specifications subject to change without notice
* see page 21-1 for specification definition
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2% Agarose, Dye-Free, 100 bp-600 bp, Marker E (external
marker)
Use Product Number:
CEF2010
Use Cassette Definition:
2% EF, Marker E
DNA Marker E Typical times to detector
Estimated Collection Times for
Minimum Size Range (Tight) Targets
Target
(bp)
150
Time to Time to Collect*
(min)
51
300
67
600
100
* Run times are affected by temperature and broad range collections.
Times should be used as an approximate guideline.
Performance Specifications*
Minimum Size Distribution as CV
Sample Recovery
100-600 bp
<8%
50 – 80%
Accuracy
<10%
Reproducibility
< 10%
Specifications subject to change without notice
* see page 21-1 for specification definitions
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