I'd appreciate any insights on how to troubleshoot my PCR.
I am trying to amplify a specific section of a gene inside the c elegans genome. This is what I've done so far:
1. DNA extraction -> yield ~2,200ng/ul
2. Taq PCR to check primers Tm. -> bright band, at right size (~1000bp) working fine for 53,54 and 55 Tms. NOTE: I did not dilute the DNA and used 1ul of original template.
3. Pfu Ultra II Fusion PCR, two Tms, 53 and 55. Diluted (100ng/ul) and not diluted templates. --> smears for samples with undiluted template, nothing for diluted samples.
4. Pfu Ultra II Fusion PCR, Tms: 49,50.1,51.1,52.1,53 undiluted template, small bright smear (near the bottom from 100 to 900bp).
5. Pfu Ultra II Fusion PCR, Tms: 48,49.2,50.2,52,52.9 diluted template (~100ng/ul) no bands at all, no smear, nothing.
Any suggestions? Is there something special I should be doing with the genomic DNA? Why do I see nothing with the diluted samples? I am thinking about trying higher Tms and/or play with the template concentration.... Please HELP!