RTpcR FOR hiv - (May/19/2004 )

I amplfied cDNA of hiv from patient's plasma, and check it by synthesis 200bp fragment from gag. It is ok. but I cannot amplfy the whole gag, envelop and nef from the cDNA. i tried the nested PCR from papers, but failed. I used the sample from Hong Kong patients. Is the mutation make me cannot do it well? How to design according to the hiv strain?

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Hi,

First of all,what is the viral load of these samples? What volume of plasma you have available? What about the quality of your RNA extraction?
I strongly recommend you to check for consensus sequences of Hong Kong or near areas in HIV Database from Los Alamos National Lab. Be sure that your primers target conserved areas...
After that,you can use a nested PCR approach. I use OneStep RT-PCR kit from Qiagen, and for the second round of PCR I use HotStarTaq from the same company.
Hope to help a little bit. Let me know.

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Hi,

First of all,what is the viral load of these samples? What volume of plasma you have available? What about the quality of your RNA extraction?
I strongly recommend you to check for consensus sequences of Hong Kong or near areas in HIV Database from Los Alamos National Lab. Be sure that your primers target conserved areas...
After that,you can use a nested PCR approach. I use OneStep RT-PCR kit from Qiagen, and for the second round of PCR I use HotStarTaq from the same company.
Hope to help a little bit. Let me know.

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