Monday, 19 November 2007

Trouble shooting for PCR

Hypotheses in order of frequency:
A. Pilot error hypothesis
B. Template dilution hypothesis
C. Temperature errors hypothesis
D. Unique template hypothesis
E. Buffer problems hypothesis
F. Bad dNTPs hypothesis
G. Bad primers hypothesis
H. Bad enzyme hypothesis
I. Bad karma hypothesis

1 comment:

praspowt said...

Pilot error hypothesis.

-most common problem with new people.
-frequently happens a couple of weeks after someone is "trained" and when they start to work independently.

- i) Other people in the lab have the same primers working on the same type of material, using the same reagents, and the same thermocycler.
- ii) You are new at PCR.
- iii) You have no experience with molecular biology.
Common causes and solutions:
- i) Miscalculation of components, especially buffer or enzyme.
- ii) Compare your mastermix receipe with others. Some early published receipes suggest using too much enzyme (say 5 units Taq to a 50 ul PCR reaction).
- iii) Did you remember to include all reagents?
- iv) You did not mix your reagent solutions prior to pipetting (usually leads to gradually worse PCR performance on a day-to-day basis).
- v) Enzyme was added to master mix prior to buffer (you killed the enzyme).
- vi) The master mix wasn`t mixed (common, but unlikely to have a consistent drastic effect; more likely to cause inconsistency).
- vii) Gross miss-measurement usually due to not knowing how to use a pipetter or a defect pipetter. Although, common to novices, it is unlikely to have a consistent drastic effect; more likely to cause inconsistency.
- viii) Oil was loaded on the agarose gel instead of the reaction contents. (If not using heated lid on PCR-block).
- ix) Oil was not added to reactions prior to running PCR. (Ignore if using heated lid on PCR-block).
- x) Wrong PCR program, or you forgot to start the program.
- xi) Wrong day of week. Go home, rest, and try again tomorrow or next week.