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In Topic: Cause of random samples failing PCR?
13 May 2013 - 05:33 PM
Ameya P, on 05 May 2013 - 11:51 PM, said:
Hi Epigeneticist,
There could be a couple of reasons why your PCR fails for a few samples.
Hope this helps.
Ameya
There could be a couple of reasons why your PCR fails for a few samples.
- SInce you are adding template DNA from various sources, their DNA concentration could be variable.
- If you are using lysis solutions for your extraction, salt remnants can also impact how well your PCR works out.
- Also, you said that you DO NOT mix your gDNA into your master mix. Well, I always mix it.
- How gDNA do you add? Porbably would be a good idea to get you pipette calibrated if you pipette volumes that are less than 2 ul
Hope this helps.
Ameya
Thanks for the response Ameya and your suggestions. My problem is due to the polymerase which is not a hot start enzyme. I was doing a poor job of keep my samples on ice during setup. I have been very careful to keep everything on ice and I haven't had any issues.
I ruled out the problems you suggested based on the following:
DNA concentration - I check my samples in duplicate and I even verified my diluted samples too.
Salt in DNA extractions - extractions are usually very good, and DNA eluates are concentrated so I have to dilute them which would dilute out the salt if I had any.
Pipettes calibration - calibrated by a certified company
Thanks again for your response! Do you have any explanation for the cause of non-specific high molecular bands (between 600-1000bp) or how to get rid of them? I have read too much template could cause this but I do not think 100ng of genomic DNA is excessive. My product is only 400bp so I decreased my extension time from 1min to 30 seconds and it got rid of the bands. Just wondering if there is another way to approach it.
