I have been trying to confirm the genotype of the uaDf5 mutant C. elegans strain, however I keep on getting undesirable results. Is this due to difficulties in preparing the crude worm lysate(i.e. DNA template) from a single worm or something is wrong with my PCR conditions.
Dirk Schmidt
since I am using a single worm, I did not measure the concentration of the DNA because the crude worm lysate is used as template DNA for PCR.By undesirable results, see the attached. It seems like there are non-specific bands in all my samples, I do not know it has something do with my PCR conditions. I used the following PCR conditions: An initial denaturation at 95°C for 5 minutes to ensure complete denaturation of template DNA. This was followed by 40 cycles of incubation steps: i) denaturation at 98°C for 5 seconds, ii) annealing at 52°C for 5 seconds, iii) extension at 72°C for 20 seconds, iv) final extension at 72°C for 1 minute and hold at 4°C.
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