Starting from leaf sample, I did DNA extraction, Mastermix preparation, PCR, run on gel till view under UV but no bands were observed, only on positive control.
First trial: Normal procedure -> no bands
Second trial: Normal procedure do together with sv -> both no bands
Third trial: Normal procedure with several dilutions -> no bands
Forth trial: Normal procedure with new extraction buffer -> no bands
Fifth trial: Procedure with ethanol precipitation -> waiting for results
1) before you go for pcr, you need to verify the integrity of the DNA. At least run gel for the DNA, confirm that there is DNA and the DNA is in good quality before proceed to pcr
2) If only positive control is working, that means that the pcr enzymes are ok, the problem may lie with your DNA (see above) or primer. You have to ensure that you use the correct primer.
Vikki Wong is right. If the pcr conditions for positive control and rest of the samples are same, then problem is most probably with your template DNA. Please send the genomic DNA gel pic with PCR gel pic.
your positive control works so pcr reagents,cycling conditions etc are all ok including the primer. A possibility is that your dna has contaminants which inhibit the pcr process so first do dilutions of your dna.Use less DNA. Sometimes less dna results in more pcr product as you dilute the inhibitors so the pcr starts to work better. Failing that do further purification of the dna or ethanol precipitate the dna and redissolve and try again. If all else fails you could design nested primers and do a second amplification of the first round product which may have worked but not well enough to see a product
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