I'm using Phenol - Chloroform - Isoamyl Alcohol solution to extract DNA from blood.
But the OD result show me the DNA concentration is too high - approximate 300ng/ul. (in the attachment)
Then I diluted in order to get the concentration around 30-50np/ul.
After that I mixed PCR with the diluted sample. And the electrophoresis result is : no reaction occured.(only show the dimer bands).
I think the problem may be in the template before diluted but I can't make sure what is it. Anyone has experience in this case?
Hi Nguyen,
1. Be sure not to get any of the bottom layer in your sample. Traces of this will give a reading and inflate your reading
http://www.biotek.com/resources/articles/240-nm-absorbance-measurement.html
Hello Nguyen,
You can directly use the DNA sample(Undiluted) in you PCR reaction.
if the amplification not occur, then there may be a problem with your Primers.
or first standardize the annealing temperature for the primers.
Hello
You can directly use the DNA sample in you PCR reaction. If your problem persists you should check your annealing temp and your primers
Dear Toan
its better to redesign your primers with appropriate guidelines.Also please standardize the annealing temperature for your primers.
Apart from the primer issues mentioned above, have you tried running some of your original DNA on a gel? It might be that it is badly degraded and broken into small fragments.
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