hello everyone,
I m trying to perform RAPD-PCR for fungal species. till date total 40+ primer i have tested but no positive result i have got, only one primer showing reproducible band again and again for all my isolate, but other primers annealing with one or two isolates only... what could be the possible reason behind it?
is this a master mix problem or a PCR cycle that is not working properly?
Try testing just one set of primers at a time. Include a good positive control and negative controls.
Test out your DNA extractions with a robust universal primer pair (e.g. actin) to make sure you actually have DNA from your samples.
Use a gradient to optimize the annealing temp for each primer pair.
When i run my genomic dna on .8% agarose gel showing good intensity of band.... Quantitative results also good in ratio and concentration ......
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