I'm currently attempting to individually clone the non-structural genes of a tick borne flavivirus, louping ill virus.
I've had success with all of the NS genes apart from NS3 (1.8kb) and NS5 (2.7kb). The melting temperature of the primers is 60-61C so I've been using an annealing temperature of 55. I'm using KOD polymserase which I've been told is a good quality enzyme.
I've tried lowering the annealing temperature to 50C and increasing the extension time to 1 minute.
I've fully sequenced the genome of the isolate I'm attempting to use, so the primers should work - additionally I also have internal primers for NS3 which I've used with the outer primers to produce two fragments which overlap by 77 bp, so I know the primers are annealing.
What could be going wrong? Any suggestions would no doubt be helpful!
Hi Jordan,
I had this problem once with amplification of 1.8kb and 2.1 kb genes of a tick species. The problem in my case was RNA integrity. short sequences of 0.5kb to 1kb were amplifying but not the longer ones. i do not know much about viral genome but i think it could be worth a try to have a fresh RNA extraction (multiple samples if possible) and cDNA prep. You also need to have an extension time between 2-3 mins as the processing rate of taq polymerase is usually 1kb/min.
Good Luck!
Hi Jordan,
as Gaurav mentioned I would increase the amplification time as 1 min is just not enough considering the average polymarization rate. Did you prepare the cDNA yourself? If so, did you used the polyT primer or the random hexamers? When preparing cDNAs form long mRNAs is better to use the random hexamers. It could also be that your PCR product is GC rich or it can form secondary structures. You could try a GC rich optimized enzyme/buffer as well as the addition of several additives to the PCR reaction such a DMSO, spermine, betaine, etc.
Hope this helps,
Ezequiel
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