User guide

24
GlobalFiler
™
Express PCR Amplification Kit User Guide
Chapter2 Perform PCR
Untreated paper substrates: prepare reactions
2
4. Calculate the volume of each component needed to prepare the reactions, using
the table below.
Note: Include additional reactions in your calculations to provide excess volume
for the loss that occurs during reagent transfers.
IMPORTANT! This kit has been optimized for a 15-µL PCR reaction volume to
overcome the PCR inhibition expected when amplifying unpurified samples.
Using a lower PCR reaction volume may reduce the ability of the kit chemistry to
generate full STR profiles.
5. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for
3 seconds and centrifuge briefly before opening the tubes or bottles.
IMPORTANT! Thawing is required only during first use of the kit. After first use,
reagents are stored at 2 to 8 °C and, therefore, do not require subsequent thawing.
Do not refreeze the reagents.
6. Pipet the required volumes of components into an appropriately sized
polypropylene tube.
7. Vortex the reaction mix for 3 seconds, then centrifuge briefly.
8. Dispense 12 µL of the reaction mix into each reaction well of a MicroAmp
®
Optical 96-Well Reaction Plate. The final volume in each well is 15 µL (reaction
mix plus Prep-n-Go™ Buffer and sample or positive control).
9. Seal the plate with MicroAmp
®
Clear Adhesive Film or MicroAmp
®
Optical
Adhesive Film.
IMPORTANT! We recommend adhesive film for plate sealing to provide a
consistent seal across all wells and prevent evaporation. Do not use caps, which
may not provide a consistent seal across all wells.
IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver
block, place a MicroAmp
®
compression pad (Part no. 4312639) on top of the plate
to additionally prevent evaporation during thermal cycling. The Veriti
®
Thermal
Cycler does not require a compression pad.
10. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge
with plate holders.
Reaction component Volume per reaction
Master Mix 6.0 µL
Primer Set 6.0 µL