Demonstration plate userguide

Demonstration plate userguide
Prime Pro 48 Demonstration Plate
FOR RESEARCH USE ONLY
Techne Part no. PRODEMO48
It is very important to promptly store the kit
contents at the temperature specified below to
ensure that they perform correctly.
Kit Contents
6
7
8
9
The Prime Pro 48 demonstration plate is shipped at
room temperature. Store it at 2°C to 8°C for up to a
month.
For storage times over one month, store the
evaluation plate at -15°C to -25°C.
10
NOTE
You will need at least 550 µl
2X SYBR Green PCR Master
Mix (total 1100ul) purchased
from a licensed supplier.
11
Pipette 20µl of the 1X qPCR master mix into
each plate well.
Remove the white backing from a plate
seal.
Place the seal on top of the plate, sticky
side down, and seal tightly using the
squeegee.
Place the sealed plate, still inside the adapter,
into the centrifuge and add another adapter
for balance. Centrifuge at 1000 g for 30
seconds. Make sure no air bubbles are
trapped at the bottom of the wells.
Recentrifuge if necessary.
Place the plate in a dark location at room
temperature and leave it for 15 minutes.
Vortex the plate for approximately 20
seconds to ensure complete solubilization of
the lyophilized reagents.
Set Up the Experiment
1
2
3
TIP
Avoid contaminating the
bottom of your plate by
holding a clean paper towel
between the plate and the
vortex head while vortexing.
Demonstration Plate
The Prime Pro 48 demonstration plate enables you
to test the performance of the Prime Pro 48 RealTime PCR System. The plate contains PCR primers
that are designed to detect and quantify an
artificial DNA sequence, with template DNA at
defined quantities or no template at all. A standard
curve with 20,000, 10,000, 5,000, 2,500, and 1,250
copies is used to quantify an unknown population
of 24 replicates.
Setup the Plate
1
2
3
Thaw 2X qPCR master mix. Pipette 550µl into
a 1.5ml tube.
Dilute master mix to 1X by pipetting 550µl
DNAse/RNAse free water into the same 1.5ml
tube. Vortex briefly to mix.
Put the evaluation plate (shown) into the
adapter and place it into the centrifuge along
with another adapter for balance. Centrifuge
at 1000 g for 30 seconds.
12
13
14
15
4
5
Turn on the netbook and launch the Prime Pro
48 Real-Time PCR software.
Click the Templates tab on the left.
Double click the demonstration plate
template file to open an experiment with
the appropriate plate layout (shown).
Edit the thermal profile as recommended by
the master mix supplier.
Click Start to start the experiment.
NOTE
Analyze the Results
To ensure homogenous resuspension of primers and
template, strictly adhere to
the time recommendations
in steps 10 and 11.
1
When the experiment finishes open Pro
Study software and analyse the data. A
plot of the standard curve appears.
2
Check results are within the following guides.
Place the sealed plate, still inside the adapter,
into the centrifuge and add another adapter
for balance. Centrifuge at
1000 g for 30 seconds.
Open the Prime Pro 48 lid by pressing the
round silver button on the front to raise its
handle, while lifting the handle from the
bottom until the Prime Pro 48 pops open.
Place the plate into the thermal block inside
your Prime Pro 48. Make sure you align the
notch on the plate with the indentation in
the back left of the block.
Close the Prime Pro 48 lid.
•
•
•
The slope should be between -3.6 and -3.1,
corresponding respectively to 90% and
110% efficiency. A slope of -3.32 indicates
100% efficiency, or an exact doubling of
template molecules in each PCR cycle.
The R2 should be approximately 0.99,
indicating a tight correlation between the
data points and the standard curve.
The standard deviation of the Cq value for
the unknown population should be less
than 0.18. Up to four outliers can be
removed from the analysis, if necessary.
The demonstration plate is in no way
intended to be used for calibration /
validation of an instrument or assay, it
is purely for demonstration purposes
only and should be treated as such.
4
5
Move the plate with the adapter onto the
Prime Pro 48 dock and turn on the dock
light.
Remove the aluminum seal from the top of the
plate, being careful not to spill any of the
plate contents. Tip: Use the squeegee to lift
the seal from the corner.
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