User guide

26
GlobalFiler
™
Express PCR Amplification Kit User Guide
Chapter2 Perform PCR
Swab substrates: prepare reactions
2
5. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate
(for accurate pipetting).
6. Transfer the sample lysate out of the 1.5 mL tubes or sample plate into tubes or
plates for storage, then discard the 1.5 mL tubes or deep-well plate containing the
swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from
the sample lysate plate before transferring the lysate.
7. Proceed to the next section to prepare the reactions or see “Store the sample
lysate” on page 27.
Prepare the
reactions
1. Add Prep-n-Go
™
Buffer (Part no. 4471406) to the control wells in the reaction
plate:
2. 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.
3. 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.
4. Pipet the required volumes of components into an appropriately sized
polypropylene tube.
5. Vortex the reaction mix for 3 seconds, then centrifuge briefly.
Well(s)
Add the following to wells of a MicroAmp
®
Optical
96-Well Reaction Plate...
Negative control 3 µL of Prep-n-Go
™
Buffer
Positive control • For 25 and 26 cycles 0 µL of Prep-n-Go
™
Buffer
• For 27 cycles 1 µL of Prep-n-Go
™
Buffer
• For 28 cycles 2 µL of Prep-n-Go
™
Buffer
Reaction component Volume per reaction
Master Mix 6.0 µL
Primer Set 6.0 µL