Should the Elucigene test not perform consistently in your laboratory, please refer to the suggestions in the tables below.
If further advice is needed, please contact our Technical Support Group by email to support@tepneldiagnostics.co.uk or by phone on +44 1235 544220. We will be happy to assist with troubleshooting.
The Normal DNA Control supplied in the kit provides valuable information to assist in the troubleshooting process. If the Normal DNA Control does not amplify consistently, initially the troubleshooting process should concentrate on the PCR and electrophoresis conditions. If the Normal DNA Control is not affected, the cause of the problem is more likely to be the samples being tested or the DNA preparation method.
– No Control or Diagnostics Bands
– Faint Control or Diagnostic Bands
– Breakthrough Banbds
– Primer-dimers seen between ~60 and 97 base pairs
– Faint Upper Control Bands
|
No Control or Diagnostic Bands
|
|
|
Possible Cause
|
Suggested Action
|
|
|
Insufficient template DNA
|
Check preparation method and solutions.
Quantify DNA and add between 10 – 50ng to each PCR reaction.
Re-prepare DNA using recommended Alkali lysis method.
|
|
PCR
|
Incorrect protocol e.g. AmpliTaq
Gold not added, DNA not added,
Incorrect program used.
|
Review protocol referring to the Instructions for Use. Repeat tests following methods described in the Instructions for Use.
|
|
|
Possible Cause
|
Suggested Action
|
|
|
Insufficient or poor quality template DNA.
|
Check preparation method and solutions. Re-prepare DNA using recommended method.
|
|
Note: ELUCIGENE products contain primers multiplexed into an optimized reaction mix. Some faint banding below the lower control band is normal, however excessive and very strong banding (i.e. as strong as the lower control band) indicates the presence of abnormal primer-primer interactions due to sub-optimal PCR reaction conditions.
|
|
|
Possible Cause
|
Suggested Action
|
|
|
Insufficient template DNA.
|
Check preparation method and solutions. Re-prepare DNA using recommended method.
|
|
DNA
|
Excess template DNA or PCR inhibitor present.
|
Dilute extracted DNA 1 in 5 and re-test.
Quantify DNA and add between 10 – 50ng to each PCR reaction.
Re-prepare DNA using recommended method.
|
|
PCR
|
Incorrect amplification program.
|
Check thermal cycler program and calibration /servicing schedule and retest.
|
| |
|
| Faint Control or Diagnostic Bands |
|
|
Possible Cause
|
Suggested Action
|
|
DNA
|
DNA poor quality or PCR inhibitor present.
|
Check preparation method, solutions and storage.
Dilute extracted DNA 1 in 5 and re-test
Re-prepare DNA using recommended method and retest.
|
|
|
Low template DNA quantity.
|
Quantify DNA and add between 10 – 50ng to each PCR reaction
Re-prepare DNA using recommended method and retest.
|
|
PCR
|
Incorrect mineral oil used or mineral oil not used. ( 0.5ml tube size thermal cyclers only)
|
Sigma light white mineral oil (M3516) is recommended. Repeat tests using Sigma light white mineral oil to prevent condensation.
|
|
PCR
|
Incorrect amplification program.
|
Check thermal cycler program and calibration /servicing schedule and retest.
|
|
PCR
|
Insufficient AmpliTaq Gold.
|
Check AmpliTaq Gold dilution calculation and volume added to reaction. Correct and retest.
|
|
GEL
|
Insufficient PCR product loaded on gel.
|
Review protocol referring to Instructions for Use. Repeat test following methods described in the Instructions for Use. Check well size - 1.5mm x 5mm combs are recommended.
|
|
GEL
|
Ethidium bromide leaching from gel during electrophoresis.
|
Gel overheated during electrophoresis, allow to cool before removing from tank for photographing.
Add ethidium bromide to gel and buffer.
|
|
GEL
|
Weak output from UV transilluminator or camera setting incorrect.
|
Check transilluminator bulbs.
Check camera settings and filters.
|
