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

Standard PCR protocol

Materials:

sterile water

10X amplification buffer with 15mM MgCl2

10 mM dNTP

50 μM oligonucleotide primer 1

50 μM oligonucleotide primer 2

5 unit/μl Taq Polymerase

template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl

mineral oil (for thermocyclers without a heated lid
1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:
10X PCR buffer 10 μl
Primer 1 1 μl
Primer 2 1 μl
dNTP 2 μl
template DNA and water 85.5 μl
Taq Polymerase 0.5 μl
2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.
3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction.
4. Place tubes in a thermal cycler preheated to 94 degrees C.
5. Run the following program:
94 degrees C 1 min
55 degrees C 1 min or annealing temperature appropriate for particular primer pair72 degrees C 1 min (if product is <500>500 bp)
for 30 cycles.
Program a final extension at 72 degrees C for 7 min.