I have used two primers (Forward and Reverse) to amplify a region from total genomic DNA. But I could see a clear band of 500 bp in the negative control as well as all other lanes. What would be the reason for this?. The expected size of the product is 750-800 bp. The length of the F and R primers were 22 and 21 respectively.
I read in a paper "Without a DNA template, primer OPA-17 produced two major (band a =
480 bp and band b = 400 bp) and one minor (band c = 670 bp) DNA products
(Figure 2A)" (Analysis of PrimerDerived, Nonspecific Amplification Products in RAPD-PCR BioTechniques 22:1071-1077 (June 1997))
Since I have experienced such an amplification in conventional PCR, I would like to know the ways that I can adopt to avoid such amplifications.
Non-specific template DNA could be the primary source for this kind of contamination. This contamination could also be due to bacterial contamination. Even few intact colonies by chance as contaminant could lead to such amplifications (see colony PCR). For this reason, normally the cocktail for PCR should be prepared under sterile conditions with clear sterile tubes!!!!
No chance for formation of such large size amplified fragment with primer dimers.
Have you got expected amplification in your samples?
You can try amplification once again with clean sterile tubes. Use sterile DDH2O (or TE suitably) for negative control.
Alternatively,
See any possibility of contamination of samples (mix up of samples in the wells) in the agarose gel while loading.
Amplified product less than or some times more than the expected size is normally happen. This needs lot of optimization (especially time of extension). You will have such kind of problem in conventional PCR!!!!!
What is the type of primers you used for amplification? Except primers such as ribosomal DNA based markers (or mitochondrial DNA primers) for genetic fingerprinting studies, one should always use cDNA for such kind of specific amplification (not the genomic DNA).
Which band has more intensity, the expectated one (750-800 pb) or the band of 500 pb?
Non-specific template DNA could be the primary source for this kind of contamination. This contamination could also be due to bacterial contamination. Even few intact colonies by chance as contaminant could lead to such amplifications (see colony PCR). For this reason, normally the cocktail for PCR should be prepared under sterile conditions with clear sterile tubes!!!!
No chance for formation of such large size amplified fragment with primer dimers.
Have you got expected amplification in your samples?
You can try amplification once again with clean sterile tubes. Use sterile DDH2O (or TE suitably) for negative control.
Alternatively,
See any possibility of contamination of samples (mix up of samples in the wells) in the agarose gel while loading.
Amplified product less than or some times more than the expected size is normally happen. This needs lot of optimization (especially time of extension). You will have such kind of problem in conventional PCR!!!!!
What is the type of primers you used for amplification? Except primers such as ribosomal DNA based markers (or mitochondrial DNA primers) for genetic fingerprinting studies, one should always use cDNA for such kind of specific amplification (not the genomic DNA).
Something wrong Mr. Rahul when you repeatedly getting amplification in negative control. I really do not understand. If you wish come to my lab with your samples I will help in this regard.
@ Prof. Janarthanan
Sir, First of all, I would like to thank you for spending your valuable time to answer my question in a detailed way.
After series of experiments, we realised that there was a contamination in the distilled water that we used for diluting DNA samples for PCR. We seriously had a doubt that one batch of our Taq was also contaminated. To clarify it, we prepared everything fresh and then, we could see the amplification of desired bands.
Hence, the problem is resolved now.
Thank you for your kind concern.
Sincerely
Dr. Rahul R. Nair
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