PCR reaction setup

PCR reactions should be set up in a dedicated laboratory adhering to clinical diagnostic laboratory rules. It may be recommended that a laminar flow cabinet with means of ultra-violet sterilization should be used. Opinion is divided on the necessity of laminar flow conditions; the operator may be lulled into a false sense of security and not heed the basic rules for setting up PCR.

1. Use aerosol resistant tips

2. Use dedicated micropipettes only. Reproducibility between runs will be affected by the use of different pipettes

3. If possible reagents, such as primer and probe stocks, and homemade master, should be prepared and aliquoted on an entirely separate site. Use the smallest aliquots that are practical. Discard empty containers; do not refill. Always discard an aliquot if contamination is suspected

4. Protect reagents for real-time PCR from light. Store aliquots in amber tubes

5. Wear gloves at all times and change frequently

6. Wear disposable gowns or keep a set of laboratory coats for PCR set-up. If different laboratories are used, change laboratory coats between areas

7. Be aware of aerosol generation. Centrifuge specimen tubes to ensure that there is no DNA/cDNA on the caps that may contaminate your gloves

8. Isolate known positive specimens from expected low level MRD specimens

9. Include NTC and NAC controls

10. Use dUTP and UNG containing master mixes

The use of plasmids as positive controls may be unavoidable; however concentrated plasmid DNA solutions contain very high numbers of target molecules. If possible plasmid DNA isolation and standard preparation should be carried out in a separate location. If possible, seal the PCR vessels containing test samples and negative controls before pipetting positive controls whether they are plasmid or cell line cDNA/DNA.

Was this article helpful?

0 0

Post a comment