Problems with cloning HIV env gene - HIV cloning (Oct/30/2007 )

hi all

i work in a hiv virology lab and we're trying to clone a novel env gene (approx 3kb), generated from RT-PCR from patient sera, we're about to give up as any colonies screened either have lost the insert or have deleted it or the vector has had fragments deleted as well. we've tried HB101/DH5alpha's, growing at 30oC, lower AMP concentrations...

if anyone has had these problems or knows of something we haven't tried all suggestions will be much appreciated.

thanks
jane

Have just this week cloned env from 2 lab strains, also amplified by RT-PCR, with TOPOXL from invitrogen (amplified by Pfu and A overhangs added after gel purification with normal Taq). What's your cloning strategy (TA, TOPO, restriction/ligation, in-fusion) , what PCR-enzymes are you using? For just env 30°C growing won't help too much, 3 kb isn't too much and env doesn't contain LTR-sequences.

hi Varius

thanks for responding! part of the problem is that we've received a PCR product of the env gene and can't seem to get it into a vector. we're tried cloning into pCRII after a taq pol step to add a-overhangs and also cloning the PCR product directly into the pCRII vector cut with EcoRV. we've also tried cutting the PCR product directly using RE sites that should be present from the oligos used to generate that PCR product. all to no avail. in addition we've tried re-ampifying the product with Phusion/Expand HiFi (using the oligos originally used for the RT-PCR) but seem to get a lot of non-specific products and not much amplification of the original product. also as this is a novel env we are a bit wary of subjecting the PCR product we have to further rounds of amplification. nothing we've tried has had much success unfortunately.

regards
Jane

For re-amplifying you would need to dilute your template 100-1000 fold, maybe then you get lucky. Are you 100% sure about your pcr product to begin with? (ran a gel of it?).

we receive the PCR product from collaborators in very limiting amounts, but we have run the product on a gel and it is not a single product, there is contamination with a smaller product(s). when re-ampifying we haven't tried 100-1000 fold dilution. what would this achieve and would you use 2-5microlitres of the diluted product in a 50microlitre PCR reaction?

thankyou for your suggestions.

regards
Jane