i was extracted theRNA form PBMCS from macaque, they were infected with SIVmac251 and put on ART therapy. i was bale to amplify actual long size band around 2920bp (env) in some samples but in some samples, the band was much shorter around 700bp. i thought due to deletion the band size was shortened. these all animals showed resistant to antiretroviral therapy. to confirm this postulate. i extracted the RNA from PBMCs collected before the ART started and surprisingly found a shorter band in all samples instead of a large band.
i am very much confused, what is happening? does anyone have an idea about or any good explanation?
Hi, Sammer,
First, did you check the extension time of your PCR? I'm sorry if I start with a very basic question, but it is a common mistake. It has to be 1 minute per 1kb. Second, I would try sequencing the 700 bp band to check what did you exactly amplified. If it is a different product, I would check in silico if your primers are not annealing in the wrong position. I would also check your annealing temperature, because if it is very low, the primers can annealing in the wrong position. But I would start sequencing the amplicon. If you have a clear and unique band, use the PCR reaction for the purification of the amplicon. Otherwise, extract the band from the gel.
Good luck!
Hi, Sammer,
First, did you check the extension time of your PCR? I'm sorry if I start with a very basic question, but it is a common mistake. It has to be 1 minute per 1kb. Second, I would try sequencing the 700 bp band to check what did you exactly amplified. If it is a different product, I would check in silico if your primers are not annealing in the wrong position. I would also check your annealing temperature, because if it is very low, the primers can annealing in the wrong position. But I would start sequencing the amplicon. If you have a clear and unique band, use the PCR reaction for the purification of the amplicon. Otherwise, extract the band from the gel.
Good luck!
as said above check your extension time ...
Try sequencing these bands and check if they show any similarity with your original product(2920) sequence...
all the best
Dear Sammer,
RIghtly pointed out by others, Sequencing would be a good way to start with identifying what is happening. Also, is there a chance that the primers also bind elsewhere in the genome? Given that 3kb is relatively not much bigger than 700bp, but is definitely more work for the polymerase, and you might be seeing something non-specific because of that. I had a similar situation, but that was because of heterozygosity of samples which is not related to your problem though.
Hope you resolve the issue.
Hi,
I was facing the same problem some time back. I increased the denaturation temperature by 2-3 degrees (not the initial denaturation but the second denaturation step which is repeated in every cycle). That fixed the problem!
Hope this works for you too.
Thanks to all.
i did sequenced the 700bp. i sequenced with two set of primers and only one work with that fragment.
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