AT rich DNA

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haf2006
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AT rich DNA

Post by haf2006 » Wed Apr 26, 2006 1:55 pm

Hi

can anyone give any advice on working with AT rich DNA. I have a vector and an insert (in a separate vector) which i have to mutate then ligate together. This is proving near impossible as i cannot get the ligation to work as i keep getting low recovery from gel extraction?

any advice on pcr methods for AT rich DNA or is there any special conditions like with GC rich?

thanks

xx :P

Ultrashogun
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Post by Ultrashogun » Sat Apr 29, 2006 9:30 am

All I know is that AT has only two H bonds so it requires less energy to take apart the strands.

oppox
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Post by oppox » Sat May 13, 2006 11:37 am

do u got the insert in a separate vector? are u going to cut it out, then ligate it into the other vector? Or are u trying to pick it up with PCR?
I have found a couple of problems with PCR on AT rich regions, dunno if they are real problems though :), I mean I dont know if I had a problem with it, but here are some stuff I changed and did.

u can lower the temp from 72 deg to 68 deg or 70, but personally I think 72 is good.

I got a blurry band on the agarose gel sometimes (when looking at the PCR product), I did a extra annealing step after the PCR reaction was done, heating it to 95 deg.

I also made a step by step cooldown in the PCR program after the last annealing step, 50 deg, 45 ,40 ,35 2 mins on each step.

As I said I dont know if this made any different because the problems wich lead me to do these changes turned out to have nothing to do with the AT-rich gene.

seaseasea
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Post by seaseasea » Tue May 16, 2006 4:27 am

there are lots of papers about PCR with high GC in PUBMED.u can search it.
hope this helps.
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