User guide

GlobalFiler

™

Express PCR Amplification Kit User Guide

Perform PCR

■ Optimize PCR cycle number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

■ Treated paper substrates: prepare reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ Untreated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ Swab substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■ Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Optimize PCR cycle number

Before using the GlobalFiler

™

Express Kit for the first time, perform a single initial

sensitivity experiment to determine the appropriate cycle number to use during

internal validation studies and operational use of the GlobalFiler

™

Express Kit. This

experiment accounts for instrument-to-instrument and sample-to-sample variations. If

you are processing multiple sample type and substrate combinations (for example,

buccal samples on treated paper and buccal samples on swabs), perform separate

sensitivity experiments for each sample type and substrate to be used for testing.

Select samples and

prepare plates

1. Select 26 of each sample+substrate type. Ensure the selected samples represent a

“typical” range of samples analyzed in your laboratory.

IMPORTANT! The number of samples recommended for this study has been

chosen to allow you to complete electrophoresis using a single 96-well plate, thus

minimizing the impact of run-to-run variation on the results. Examples of PCR

and electrophoresis plate layouts are provided on page 73.

2. Prepare the samples and the reactions as described in the appropriate protocols

later in this chapter. Prepare sufficient PCR reagents to complete amplification of

three replicate plates.

3. Create three identical PCR plates (see page 73 for a suggested plate layout).

4. Amplify each plate using a different cycle number to determine the optimum

conditions for use in your laboratory. Suggested cycle numbers for different

sample type and substrate combinations are listed below.

Sample

type

Substrate

Treated paper Untreated paper Swab

Blood 25, 26, 27 cycles 25, 26, 27 cycles N/A

Buccal 26, 27, 28 cycles 26, 27, 28 cycles 25, 26, 27 cycles