User guide
66
GlobalFiler
™
Express PCR Amplification Kit User Guide
Appendix A Troubleshooting
A
More than expected
number of alleles
present at a locus
Presence of exogenous DNA Use appropriate techniques to avoid introducing
foreign DNA during laboratory handling.
Amplification of stutter product
(–1 repeat unit position)
See “Experiments and Results” on page 63.
Incomplete 3´ A base addition (n-1 nt
position)
See “Experiments and Results” on page 63. Be
sure to include the final extension step of 60°C for
5 minutes in the PCR.
Signal exceeds dynamic range of
instrument (off-scale data)
Ensure cycle number is optimized according to
instructions on page 19. Repeat PCR amplification
using fewer PCR cycles or use your laboratory’s
SOP to analyze off-scale data.
Poor spectral separation (bad matrix) Follow the steps for creating a spectral file.
Confirm that Filter Set J6 modules are installed
and used for analysis.
Contamination carried over from the
disc punching tool
Clean the disc punching tool thoroughly. If
necessary, include a blank punch step in between
the sample punches.
Incomplete denaturation of double
stranded DNA
Use recommended amount of Hi-Di
™
Formamide
and perform heat denaturation step according to
the instructions in Chapter3, “Perform
Electrophoresis”.
Some but not all loci
visible on
electropherogram of
DNA Test Samples
Disc size used in the amplification
reaction was greater than 1.2 mm
Repeat amplification using a use 1.2 mm punch
size.
Insufficient volume of swab lysate
added to the reaction
Repeat amplification using the recommended
lysate input volume.
Less than 15 µL of PCR reaction
volume was used
Repeat amplification using the recommended PCR
reaction volume of 15 µL.
STR profiles contain
many off-scale alleles
PCR cycle number was too high Perform sensitivity experiment (page 19) to
determine the optimal PCR cycle number based on
the sample type.
For blood samples: Too much liquid
blood was spotted onto paper
substrate
Spot <100 µL of liquid blood per sample area.
Data collected on the
3730 instrument with
POP-7™ polymer fails
sizing
The 60 bp size-standard peak is
occasionally obscured by the primer
peak
• Re-inject samples that fail to recognize the 60
base-pair peak.
• Use the 80 to 460 bp size-standard definition
after performing appropriate validation studies
(as a general rule, the 60 base-pair peak is not
required for accurate fragment sizing using the
3rd Order Least Squares sizing method).
For more information, refer to the GeneMapper®
ID-X Software Version 1.4 User Bulletin
(Pub. no. 4477684 Rev. B), “Known issues: 3730
DNA Analyzer sizing failures”.
Observation Possible causes Recommended actions