Ready-To-Run electrophoresis system

user manual
electrophoresis system
Ready-To-Run
Electrophoresis System
a compact electrophoresis system for
high-throughput PCR screening
um 80-6468-74/Rev. AO/06-00
Page finder
Unpacking
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Specifications
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Important safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Components of the Ready-To-Run electrophoresis system
Operating instructions
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Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Care and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Customer service information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
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• pii
• unpacking
Unpacking
Unpack all packages carefully and compare contents with the packing
list, making sure all items arrived. If any part is missing, contact your
local Amersham Biosciences sales office. Inspect all components
for damage that may have occurred while the unit was in transit. If any
part appears damaged, contact the carrier immediately. Be sure to keep
all packing material for damage claims or to use should it become
necessary to return the unit.
Specifications
Selectable parameters:
Voltage
120 VDC, 90 VDC, 60 VDC
(±10% or ±10 V, whichever is larger)
Time
0–99 min, 1 min resolution
User interface
Two-digit LED display, membrane keypad
Power requirements:
Frequency
47–63 Hz
Line voltage
115/230 VAC
Power consumption
50 W maximum (with up to three satellite units)
Dimensions
16.5 × 15 × 16.5 cm (h × w × d)
(each base and satellite unit)
Weight
2.0 kg (base unit), 1.6 kg (each satellite unit)
Satellite units
Up to three satellite units can be connected to a base unit
Operating environment
Indoor use
Temperature
5–40 °C
Relative humidity
80% maximum up to 31 °C,
decreasing linearly to 50% at 40 °C
Safety specifications:
Safety
UL3101-1: 10/93, CSA 22.2 No. 1010, EN61010-1
Emissions
EN61326
Listed by ETL Testing Lab
CE Marked
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ready-to-run electrophoresis system • important safety information
Important safety information
• Always disconnect the power cord before servicing.
• Plug the instrument into a properly grounded outlet.
• Ready-To-Run™ gels contain ethidium bromide, a known mutagen.
Introduction
Rapid PCR screening
The Ready-To-Run electrophoresis system simplifies screening the large
numbers of PCR products generated for applications such as high-throughput
sequencing, cDNA array production, genotyping, and library screening. The
Ready-To-Run system consists of a separation unit with a built-in power
supply, precast agarose gels, and a Loading Guide to facilitate sample loading.
A single Ready-To-Run Separation Unit separates up to 96 samples in
5 min or less. For even greater throughput, one to three additional optional
Ready-To-Run Satellite Units can be added to the system to allow as many
as 384 samples to be run simultaneously.
Microtitre format precast gels
In addition to high throughput, the Ready-To-Run system also features
the convenience of high-quality precast agarose gels that maintain both
the dimensions and sample spacing of standard 96-well microtitre plates.
Conformity to sample spacing in microtitre plates simplifies sample loading and analysis of results. The straightforward correspondence between
the position of a sample in the microtitre plate and its position on a gel
also reduces the chance of introducing errors in loading and analysis.
The gels are ready to use out of the package. The buffer required for electrophoresis is integral to the gel itself, eliminating any need for preparing
and handling liquid buffer. Ethidium bromide is included in the gel, so
the gel is ready for analysis as soon as electrophoresis is complete. Where
greater separation distance between bands is required, a 48-well gel is
also available. Both gel formats include two additional wells, one at
either end of each sample row, for molecular weight standards (Fig 3).
Simplified sample loading
The Loading Guide facilitates sample loading by positioning pipette tips
within the sample well and preventing puncturing of the gel. It can be
used with 1-, 8-, or 12-channel pipettors. Two versions of the Loading
Guide are available to accommodate tips from most major manufacturers
(see Fig 4).
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• simple operation
Simple operation
With straightforward time and voltage selection, the Ready-To-Run
system incorporates a user interface that is both simple to use and sufficiently flexible to meet most needs. After loading the gel and placing it on
the instrument, simply close the safety lid, select a voltage setting (60, 90,
or 120 V), the run duration (1–99 min in 1 min increments), and press
the start/pause key. Power to the gel is terminated automatically at the
end of the run. At the user’s option, a buzzer indicates completion of the
run. A run may be temporarily interrupted to examine the gel before the
entered time has elapsed by pressing the start/pause key again. After
returning the gel to the unit, press the start/pause key to resume the run
from the time at which it was paused. The stop key terminates electrophoresis and resets the timer to the last entered value.
Components of the Ready-To-Run
electrophoresis system
The features of the three components of the Ready-To-Run system are
listed below.
Separation unit
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Fig 1a. Ready-To-Run Separation Unit.
Built-in power supply
Timer (0–99 min in 1 min increments)
Three voltage selections: 60 V, 90 V, and 120 V
Optional buzzer indicates end of run
Once a run has begun, it may be paused or terminated
Accepts up to three satellite units
Fig 1b. Ready-To-Run keypad.
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ready-to-run electrophoresis system • components of the ready-to-run electrophoresis system
Ready-To-Run Satellite Unit
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Optional accessory module for use with a base separation unit
Timer (0–99 min in 1 min increments)
Three voltage selections: 60 V, 90 V, and 120 V
Optional buzzer indicates end of run
Once a run has begun, it may be paused or terminated
Ready-To-Run agarose gels
• 1.2% agarose in 1X TBE, 0.5 µg/ml ethidium bromide in a UV-transparent
disposable cassette
• Specifically designed for use with Ready-To-Run Separation Units
Fig 2. Ready-To-Run Separation Unit and
Satellite Units.
• Standard microtitre-plate dimensions simplify loading and analysis of
PCR products from a 96-well plate
• Two gel sizes are available: 96 sample wells (plus 16 reference wells)
or 48 sample wells (plus 8 reference wells)
• Sample well volume: 10 µl
• Reference well volume: 6 µl
Loading Guide
• Fits over gel cassette, with holes that keep tips from puncturing the gel
• Simplifies loading large numbers of samples by guiding pipette tips through
tapered holes into sample wells of the gel below
• Reduces “loading fatigue” of arm when loading large numbers of samples
• Works with both 8- and 12-channel pipettors
• Two versions available to accommodate 10 µl tips from numerous
manufacturers:
Fig 3. Ready-To-Run agarose gels.
Style A:
Continental Lab Products (#2340)
Fisher (#21-276A)
Matrix (#7611)
USA Scientific (#11113800)
VWR (#53509-060)
Style B:
CLP (#2142)
Costar (#4830)
Eppendorf (#2235153-2)
Fisher (#21-197-2E)
VWR (#53511-680)
If using a tip not listed above, confirm that
the end of the tip extends 2.5–3 mm (but not
more than 3 mm) below the bottom surface
of the Loading Guide.
Fig 5. Tip depth.
Fig 4. Pipette tip styles.
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• operating instructions
Operating instructions
Sample preparation
Ready-To-Run gels are designed to be used without liquid buffer. As
such, certain precautions must be taken for optimum results. First, the gel
must not be allowed to dry out before use. Do not open the gel package
until you are ready to load samples, and run the gel immediately after
loading. Second, fill all sample wells completely and reference wells with
sample and/or buffer (8.5–10 µl and 5–6 µl respectively) to ensure that
the electric field is uniform throughout the gel. Otherwise, local distortions of the field around underfilled wells will cause distortion of bands.
Chevron (^)-shaped bands indicate under-filled wells.
Either of the two methods for loading samples described below will
ensure that the sample wells are full and the electric field is uniform. The
first method is the same as used for a conventional submarine gel. Each
sample is premixed with an appropriate volume of buffer to completely
fill sample well. The second method, the sample wells are prefilled with
buffer, followed by the addition of PCR samples directly from the reaction
plate. This method avoids the premixing step of the first method and may
be preferred where throughput is a priority.
Method 1:
Mix 1 µl (1/25–1/100 of PCR reaction volume) of sample with 9 µl of 1X
loading dye or TBE in a clean microtitre plate. Load at least 8.5 µl of this
mixture into each well. A useful variation of this method is to preload the
wells with 1X TBE (e.g. 5 µl) and then add a volume of diluted sample
(e.g. 3.5–5 µl ) to bring the total volume in the wells to 8.5–10 µl.
Method 2:
Fill the wells with TBE by flooding the entire surface of the gel with
approximately 5 ml of 1X TBE or distilled water and then draining the
excess liquid by tilting the cassette. Use a labwipe to absorb remaining
fluid at the corner. Wells should remain at least 85% full. Samples
(approximately 0.5–1 µl) can be loaded directly from microtitre plates
without adding loading dye. Standards can be loaded in the same manner.
We have found that samples taken directly from PCR reactions or from
most commercial preparations of molecular weight standards are sufficiently dense that no additional density agent is required to keep samples
in the wells. Furthermore, the sample mixes with the buffer in the well
sufficiently rapidly to produce uniform bands.
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ready-to-run electrophoresis system • operating instructions
Notes:
• Be sure that sample wells contain at least 8.5 µl of sample/buffer mixture
before beginning a run. Underfilled wells produce distorted band shapes.
Preferably any wells on a gel that will not contain a DNA sample should also be
filled, although this is not essential.
• Generally, 0.5–1 µl of a PCR reaction can be used, but as little as 1/200 of a
PCR reaction may be sufficient. For optimal resolution of DNA markers, adjust
the concentration to 5–10 ng/band in 5 µl. Ready-To-Run gels are thinner than
most agarose gels, so less sample is required.
• Standard loading-dye recipes containing glycerol, sucrose, or Ficoll™ can be
used. We recommend that the concentration of any tracking dye (e.g.
bromophenol blue or xylene cyanol) be less than 0.025%, as excess dye may
obscure some bands.
Gel preparation and sample loading
1
Remove the Ready-To-Run gel cassette from the foil package. Remove the two
tape strips from the bottom of the gel cassette, then remove the plastic sheet
from the top surface of the gel.
2
Place gel cassette on bench (not on electrophoresis unit) with the + sign facing
you. With the notch on the Loading Guide toward the upper-left corner and the
letter A or B at the lower right, place the Loading Guide over the gel.
3
Hold the guide in place and insert pipette tips straight down through the holes
and into the wells. Do not force tips into the holes. Expel sample. Hold the guide
in place on the gel cassette when removing the tips from the guide (see Fig 6).
4
Fig 6. Correct loading procedure.
Remove guide from the gel. Run the gel immediately after loading samples to
avoid diffusion and drying artifacts.
Notes:
• Remove the tape and protective cover sheet just prior to use. Once gel has been
removed from the package, use as soon as possible. Otherwise, gel may dry out
and affect separation results.
• Do not load gel on the separation unit. Place cassette on bench while loading
samples to avoid damaging the gel.
• See illustrations and list of tips above to determine which version of the
Loading Guide to use (A or B).
• If tips extend > 3 mm past the bottom of the guide plate, the guide plate will
not prevent the tips from damaging the well.
• Use of excess force when inserting pipette tips may cause the tips to be caught
by the guide plate upon removal.
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• p6
• running the gel
Running the gel
1
Turn on the separation unit with the power switch on the rear of the unit.
2
Select voltage (60, 90, or 120 V) by pushing the V key (on the keypad) until
desired voltage is illuminated.
3
Select time in minutes by using the + and – arrows. As a rule of thumb, at
120 V and 5 min, a 200 bp DNA will migrate to just above the next row of wells
on a 96-well gel. At 120 V, it will take a 200 bp DNA about 10 min
to migrate to the next row of wells on a 48-well gel. The rate of migration is
proportional to voltage, so double these times when the voltage is set at 60.
Fig 7. Orientation of Gel Cassette and
Separation Unit.
4
After loading the samples as described above, open the lid by pushing down in the
middle of the front of the lid then gently place the gel onto the platform of the
separation unit. It is not necessary to press the gel down on the electrodes.
The cathode (–) and anode (+) are indicated on the gel platform.
5
Close lid and press start/pause. Power to the gel shuts off automatically when the
set time has elapsed; the display flashes “00” for 1 min to indicate that the run
was completed.
6
Open the lid and remove the gel cassette from the separation unit.
Notes:
• Do not force the gel cassette down onto the unit. The weight of the gel cassette
is sufficient to maintain contact between the gel and the electrodes.
• To Pause a run: Push the start/pause key to shut off power to the gel and halt
the timer; push the start/pause key again to restart power to the gel and resume
counting down from the time at which the run was paused.
• To Stop a run: Push the stop key during electrophoresis to shut off power to the
gel and reset the timer to the last entered value.
• The optional buzzer to indicate completion of a run is toggled on and off by
simultaneously pressing the + and stop keys.
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ready-to-run electrophoresis system • visualizing results
Visualizing results
Visualize results by placing the gel cassette on a UV transilluminator
(302 nm). Results can be documented with film-based or digital camera
systems, or with laser-scanning systems designed to detect ethidium
bromide–stained DNA. Note, however, that some video documentation
systems may not have sufficient resolution to display or record all of the
useful information in a Ready-To-Run gel.
Note: Refer to local regulations for proper disposal of ethidium bromidecontaining gels.
Attaching satellite separation units
Ready-To-Run satellite units are optional modules that can be attached
to a Ready-To-Run Separation Unit where greater throughput is desired.
All the keypad functions available in the Ready-To-Run Separation Unit
(voltage and time selection, start/pause, and stop) are available to each
attached satellite unit. Satellite units do not contain a power supply and
will not function except when attached to a separation unit. One, two, or
three satellite units may be attached to a separation unit (connection of
more than three satellite units with a single separation unit will result in
an “E4”, and power will not be delivered to the fourth unit). So long as
the power switch of the base separation unit has been turned on, each
satellite unit functions autonomously, regardless of whether the base
separation unit is actually running a gel.
To attach a satellite unit to a base separation unit, or to attach two
satellite units together, follow the steps below and refer to Fig 8.
Note: When viewed from the front, the base separation unit must always
be the left-most module in any combination of units.
1
Lay the units to be attached next to one another on the bench, facing down
with the bottom of the units facing toward you.
2
Unscrew the four screws shown in Fig 8.
3
Fasten the communication cable between the units as shown, making sure
the connector is fully seated.
Fig 8. Attach Satellite Separation Units. Refer to
steps 1–5 at the right.
4
Insert the tabs in the connector bracket into the corresponding slots in the
bottom of the two units.
5
Fasten the bracket to the two units with the four screws provided.
Repeat steps 2–5 if additional units are to be added, remembering that the base
separation unit must always be the left-most module in any combination of units.
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• troubleshooting
Troubleshooting
Error messages.
E1 over-current condition. Check that no buffer or other conductive material is causing a short circuit
under the cassette between the electrodes.
E2 safety lid is open. Close lid.
E3 open-circuit condition. Check that tape strips on gel have been removed and that gel is positioned
on the electrodes properly.
E4 more than three satellite units are attached to the base separation unit. Remove extra unit(s).
Can’t see sample bands or bands look faint.
Too much dye loaded with sample.
Reduce dye in loading buffer (< 0.025%).
Not enough DNA loaded.
Load more DNA.
Samples are smeared.
Samples not properly loaded in wells.
Hold pipettor perpendicular to Loading Guide while loading samples.
Gel dried out before electrophoresis.
Run gel immediately after loading samples. Do not use expired gels.
Too much DNA loaded.
Dilute sample further before loading.
Sample contains contaminants.
Clean up PCR template or final product.
^
Chevron ( )-shaped bands, sometimes streaked.
Wells not full.
Load 7.5–8 µl into sample wells, 4.5 –5 µl into reference wells.
Care and maintenance
• After use, wipe away any liquid that has accumulated at the cathodic
electrode. To remove salt buildup on electrodes, clean with mild detergent and water. Do not immerse unit in water.
• Never use abrasive cleaners, solvents, or chloroform on any part.
Customer service information
Technical service and repair
Important! Request a copy of the Amersham
Biosciences “Contamination Clearance
Certificate” form before returning the item.
No items can be accepted for servicing or
return unless this form is properly completed.
Amersham Biosciences offers complete technical support for all
our products. If you have any questions about how to use this product, or
would like to arrange to repair it, please call or fax your local Amersham
Biosciences representative. A two-digit number identifying the
installed firmware version appears for several seconds in the 7-segment
LED display during the self-test sequence that occurs when the Ready-ToGo Separation Unit is turned on. If you experience problems with the
Ready-To-Go Separation Unit that you suspect are related to firmware,
please provide this version number to your Amersham Biosciences
representative.
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ready-to-run electrophoresis system • ordering information
Ordering information
Ready-To-Run Separation Unit
80-6460-95
Ready-To-Run Satellite Unit
80-6461-14
Ready-To-Run Agarose Gel, 1.2% agarose, 96-well (10 gels)
80-6461-33
Ready-To-Run Agarose Gel, 1.2% agarose, 48-well (10 gels)
80-6461-71
Ready-To-Run Loading Guides
80-6462-09
Associated products
Ready-To-Go™ PCR Beads (0.5 ml tubes)
100 reactions
27-9555-01
Ready-To-Go PCR Beads (0.2 ml tubes/plate)
96 reactions
27-9553-01
Ready-To-Go PCR Beads (0.2 ml tubes/plate)
5 × 96 reactions
27-9553-02
Ready-To-Go PCR Beads (0.2 ml hinged tube with cap)
96 reactions
27-9556-01
Ready-To-Go PCR Beads (0.2 ml hinged tube with cap)
48 reactions
27-9556-02
Ready-To-Go RT-PCR Beads (0.2 ml hinged tube with cap)
96 reactions
27-9259-01
Ready-To-Go RT-PCR Beads (0.2 ml hinged tube with cap)
48 reactions
27-9259-02
Taq DNA Polymerase (cloned)
250 units
T0303Z
Taq DNA Polymerase (Thermus aquaticus)
250 units
27-0799-01
dNTP Set, 100 mM solution
4 × 25 umol
27-2035-01
PCR nucleotide mix
500 ul
US77119
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Ficoll, Hoefer, Ready-To-Go, and Ready-To-Run are
trademarks of Amersham Biosciences Limited
or its subsidiaries.
The Polymerase Chain Reaction (PCR) is covered
by patents owned by Roche Molecular Systems and
F Hoffman-La Roche Ltd. A license to use the PCR
process for certain research and development activities accompanies the purchase of certain reagents
from licensed suppliers such as Amersham Biosciences
Limited and its affiliates when used in
conjunction with an authorized thermal cycler.
Amersham and Amersham Biosciences is a trademark of Amersham plc.
Polaroid is a trademark of Polaroid (UK) Ltd.
© 2000 Amersham Biosciences Inc.
All rights reserved.
All goods and services are sold subject to the terms
and conditions of sale of the company within the
Amersham Biosciences group that supplies
them. A copy of these terms and conditions is
available on request.
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