Hai, I am trying to isolate DNA from frozen and fresh fish samples for PCR.
For fresh samples i am getting a clear DNA band in the agarose gel, but in the case of frozen fish i am getting smear. The same problem occur in the PCR product also. How to get the good quality DNA from Frozen fish.
Here i attach those DNA and PCR product. freash samples give clear bands and frozen fish give smear.
Please give the suggestion to get rid of this problem.
Thank you
Please calrify me, is the first gel pic showing genomic DNA. If yes then how come you got two bands?
Frozen at what temperature? -80 is best for a few months.
But after u start DNA extraction you need to CI wash many times and add RNAse at incubation step.
Dear Mr. Siva Bala
The appearance of smear after running extracted DNA on gel indicates that the DNA is degraded either from start of extraction or is degraded during the DNA extraction step. I will recommend you few things.
1) Add RNAses in your extraction procedure.
2) Switch to an efficient DNA extraction protocol i-e use commercial Kit.
3) Check reagents of your DNA extraction protocol for expiry.
The second image as you have uploaded dear Siva Bala indicates the PCR product run inspite of just extracted DNA run, if you are getting a smear in your PCR product. Then simply increase the annealing temperature of your primers the smear will disappear.
Hope your would have got the answer.
Thanks
Thank you Dear Ovais, I have not used RNAse in the previous extraction, i will try with RNAse.
Thank you again for your suggestion
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