I work with HCV-4, and I want to amplify the full lengh of the NS5B gene for the HCV4 type. Could anyone share the primer sequences and the PCR program?
hi, I think that I can help,becouse my tezes,because my theses was for full genome hcv 1a.
Hi, Dr. Murad, how do you do for full genome, uses many primers or one primers. I amvery interesting in to have full lenght of the HCV.
Despite appearing counter intuitive, it is more efficient to use random primers in the RT step prior to using specific primers in the PCR. Make sure your PCR primers are designed to ensure the 3' nucleotide is not in position 3 of a codon.
Dear Dawood Mousavi, i hope to profit from your experience, so i confirmed by PCR that i have a HCV-4 in my sample, and i selected by virtual PCR the NS5B full length (1776 bp), so i want to isolate this gene.
Dear Dr Eric Gowans, yes i already used the random primers for RT step, i selected the primers to cover full length NS5B gene for the genotype HCV4
Dear Dr Fernando Puerto, for the first step, i used degenerate primers to confirme that i have a HCV4 genotype
Dear Dr Murad, me too used degenerate primers of the FERMETAS, but, I wat use one only primer for all HCV genome, do you have papers about this?
I think the best way is making cDNA with random primers. Its concentration is very important. For the size of 1800 bp I think 120 ng/ul is good. Since with higher conc. of random primers you obtain shorter fragments.
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