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NESTED PCR - (Mar/22/2014 )

I am using semi nested PCR for amplification of ITS region of a nematode. For both rounds of PCR, I am using same positive sample. After first run, I dilute positive along with all other samples. In my test samples, DNA quantity is too low to detect on gel after first round. After second run, some test samples show positive results. But my positive show double band. That makes sense. Should I run new positive for both runs?

-Soni Khan-

Please reply my answer.

-Soni Khan-



What exactly do you mean when you say double band? Two bands close to each other or a band that you are looking for and another unspecific band in the lane. 


Please upload a picture, if possible along with some information about the primers, so that we can help 

-Ameya P-

For my first round of PCR, I am using primers for amplification of whole ITS2 region (approximately 350bp) for this nematode . After diluting these samples, I use second set of primers to amplify almost 180 bp region. but my sample which I run as a positve shows two bands on final gel one for 350bp and second for 18bp?

-Soni Khan-

sorry  it is 180bp not 18bp:)

-Soni Khan-

Well, it appears your positive control amplifies well with the initial primers, giving lots of product. You are probably not diluting the pcr product sufficiently in the second round, so the product of the second round has both the product of the initial round (350 bp) and of the second round (180 bp) visible on the gel. The test samples likely do not amplify well enough with first round primers to show a gel band. This is not surprising, depending on the source of your control reaction DNA and the amount of dilution you do for that sample between initial and final PCR reactions.


Thank you.

-Soni Khan-