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DESPERATE FOR HELP!

RNA RT-PCR

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#1 Dr. Happy

Dr. Happy

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Posted 09 March 2012 - 09:47 AM

hey there
i'm trying to isolate the HVR of HCV-E2..i've extracted RNA from patients' sera using biozol followed by RT-PCR and then first PCR followed by a second PCR using nested primers..i use to have good results before but i failed to clone my fragment Posted Image..
these days i've purchased a new cloning kit and tried to amplify my fragment as a fresh start and i followed the exact protocol i've optimized over years but i got no results!
to be honest the biozol i'm using is expired but well maintained at +4C and gave me good results just two months ago...it seems O.K and i even i measured it's pH and found it about 4.4...
i changed every single thing starting the 0.2 PCR tubes, RT-enzyme, water, dNTPs and the Taq polymerase i use for first and second PCR and all i've got is primer dimer sometimes with a smear..
what in god's name is going wrong??? RNA extraction?? or my PCR components???!! Posted Image
if anyone can help me i'll really appreciate it..
best regards for u all
happy





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