User guide

20
GlobalFiler
™
Express PCR Amplification Kit User Guide
Chapter2 Perform PCR
Treated paper substrates: prepare reactions
2
Note: Our testing has not included blood samples on swab substrates. This
sample type is not frequently used for the collection of database or casework
reference samples.
Note: To minimize the effect of instrument-to-instrument variation, use the same
thermal cycler to amplify all three plates. To maximize result quality, prepare and
amplify Plate 1 then repeat for Plates 2 and 3. Do not prepare all three plates
simultaneously.
Determine
optimum
conditions
1. Run the PCR products on the appropriate CE platform using the recommended
protocol; see Chapter 3, “Perform Electrophoresis” on page 29.
2. Based on the results of the sensitivity study, select the appropriate PCR cycle
number for future experiments.
Our studies indicate the optimum PCR cycle number should generate profiles with the
following heterozygote peak heights, with no instances of allelic dropout and minimal
occurrence of off-scale allele peaks:
The GlobalFiler
™
Express Kit is optimized to amplify unpurified:
• Single-source blood samples on treated paper or untreated paper
• Buccal samples on treated paper, untreated paper, or swabs
When amplifying single-source, unpurified samples using the GlobalFiler
™
Express
Kit, you should expect to see greater variation in peak height from sample to sample
than is expected with purified samples. Careful optimization of the cycle number will
help to minimize this variation.
Treated paper substrates: prepare reactions
Sample prep
guidelines
• Do not add water to the wells on the reaction plate before adding the punches. If
your laboratory is experiencing static issues with the paper discs, you may
prepare and dispense the 15 µL reaction mix into the wells of the reaction plate
before adding the punches.
• Make the punch as close as possible to the center of the sample to ensure
optimum peak intensity. Increasing the size of the punch may cause inhibition
during PCR amplification.
• For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card,
hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press
and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction
plate.
• For automated punching: Refer to the User Guide of your automated or semi-
automated disc punch instrument for proper guidance.
Instrument Heterozygous peak height
3500 Series 3000–12,000 RFU
3130 Series 1000–3000 RFU
3730 3000–12,000 RFU