Need help for PCR for AT rich gene - (Dec/10/2011 )
Dear All,
I am new to molecular biology and so to this forum, writing for the first time in hope of some help regarding amplification of genes i am working.
I have recently started my work on 3 genes from moraxella catarrahlis organism. I have designed primers for three genes and all of them are around 700bp long and rich in AT. GC is very less, so long stretch of As and Ts are there. If i take only gene in consideration then designing primers was difficult because of less GC content, so i have gone downstream at 5' and upstream at 3' end.
Primers look good now. But i have tried so many conditions, dec and increasing Mg2+ ion concentration, changing primer concentration, changing different annealing time etc,, but in none of them amplification is seen, even no non-specification is observed, gel looks so blank. I have tried hot start and hot start in combination with touch down PCR.
Also recently I read a paper for AT rich gene amplfication suggesting to lower down extention temperature. I have tried a gradient of reaction from extention temperature from 70 to 60 degrees in both during cycle and final extention. Also increased extention time. But again no amplification is there.
I will be delighted if someone can suggest something that may b useful and beneficial.
Thanks and Regards
I have done PCR through poly AT regions as long as 30 bp. You probably read the Su paper, and indeed the extension temperature is the key. In my case, I could not get the PCR to work with an extension temperature higher than about 63 degrees. I doubled the normal extension time, but that was probably more than necessary. You might try out some of the newer enzymes such as Phusion that have DNA binding motifs to hold the enzyme on the strand.
Often it is suggested to use betaine to make a PCR work in AT-rich regions. Also the companies say this about their additives (Q-solution (= betaine), PCRx Enhancer Solution etc). No idea if it's true, but an attempt might be useful.
phage434 on Sat Dec 10 16:06:02 2011 said:
I have done PCR through poly AT regions as long as 30 bp. You probably read the Su paper, and indeed the extension temperature is the key. In my case, I could not get the PCR to work with an extension temperature higher than about 63 degrees. I doubled the normal extension time, but that was probably more than necessary. You might try out some of the newer enzymes such as Phusion that have DNA binding motifs to hold the enzyme on the strand.
Thanks a lot Phage434 and Hobglobin for your replies. I tried reducing the extension temperature, I tried gradient of extension temperature from 70 to 60 degrees but couldn't get anything. I have a doubt whether extension temperature is decreased during the cycles or the final extension is also decreased and kept same and set according to the the cycling temperature.
It will be highly appreciable if you can provide a protocol you have used, it will be of great help.
And i will also try to use some additives as suggested by Hobglobin, but a protocol will be of great help again.
Again thanks a lot for your help.
