Hello! I am having difficulty amplifying the coding sequence of RSV F protein.
The protocol I have used is a sfollows: I infect HEp-2 cells with the virus, then when CPE is evident I collect the cells and use the RNeasy kit from Qiagen to purify the RNA with good yields. Then I have tried to do the RT with supercript IV or Protoscript (NEB), using gene specific primers, oligodT or random primers. And finally I have tried PCR with different concentrations of template, DMSO, Mg2+, temperatures.
Does anyone have any piece of advise?
Peter Yang
After RT, you can add RNAse to digest RNA and then isolate the cDNA from the enzymes. If you have good DNA yield, then you are probably having a problem with PCR. Try doing PCR with the isolated DNA instead.
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