Does anyone know why after extracting Cryptosporidium DNA from feacal samples, the first PCR works well but when I need to run another PCR from the same extracted DNA, it does not work on gel picture although the positive control was working. I am using bioline kit but this issue happened many times for me. I hope to find help from you.
Regards,
Does Cryptosporidium DNA has introns? If so, check if your primers bind to the exon-exon junctions or to two different exons which are separated by introns. I am assuming here that you are using cDNA as a positive control.
thanks Mohsen for your co-operation, I'm really using cDNA as a positive control but I'm predicting that Cryptosporidium DNA has intron since my PCR approach targeting 18s rRNA. in addition, most of eukaryotic protozoan DNA have got introns within their genome
Regarding my primers, I think they bind with the exon position because the DNA was working well in the first time after extraction but when I return back to amplify another products from the same DNA, no band was appearing on the gel (no amplification )
So are you saying the first primer set worked, but the second primer set did not work? Every primer set has different PCR parameters even with the same DNA.
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