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