I wana amplify a rigon of about 900 bp of HBV genome. my samples collected 2-3 years ago and saved at -80. PCR conditon has optimized with fress samples but unfurtunatly dose not work seccessfully with my samples. anybody has a suggestion to me ?
Dear Abbas ali Husseini,
Replication competence is highly sensitive to mutations. This fact could lead many of the replication-competent HBV genomes from a clinical specimen to keep their replication and antigen expression phenotypes even after extensive amplification. I enclose a similar study to yours to my answer. May be it could help you about your problem.
Marcellin.
Did you tried to test the integrity of your DNA on a gel?. If yes, there many other reasons that can result on this but you need to write down your protocol
the first step you should do is to test the quality and quantity of DNA then check your PCR conditions
I agreed with Mohammed Alhomidi, you should first quantify and load the template of your sample in range from 10 ng-100 ng per PCR reaction.
Please recheck the annealing temperature by running Ta gradient PCR and choose the Ta with maximum amplification and single peak in agarose gel.
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