>To anyone on the net who knows:
>> I have been attempting to PCR a fragment on the Drosophila
>Simulans TRA gene with almost no luck. My primers are both 17 bases long and
>they span a segment of about 800 bases. I've tried many different annealing
>temperatures from 53 - 65 degrees Celsis, and have also tried different
>concentrations of genomic DNA, I've run 25 cycles, 30 cycles, and 35 cycles,
>and I still can't get the damn thing to work. Does anyone know anything I
>don't know about genomic fly DNA and the effective PCR of these DNA?
>> Please respond. My email address is: jalin at ucsd.edu Thanks.
This happened to us once with a D. simulans gene as well. The problem is
more likely the primers than the DNA. PCR is very robust and the amount of
DNA and quality of DNA can be very flexible. There are two possible
problems with primers: 1. mismatch of your primer with your template DNA;
2. primers form dimers and/or hairpins. I suggest that you try different
sets of primers. A program Oligo 4.0 worked very well for us in search for
PCR and sequencing primers. By the way, we use PureGene kit (very simple)
to isolate gDNA from single flies, and it works consistently.
Department of Ecology & Evolution
The University of Chicago
1101 E. 57th Street