Help in PCR: Regarding amplification of AT rich gene

[shivu]

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

[m37786]

dear shivu
can you try with ready PCR master mix such as Promega M7122
mahmoud manama

[jonoave]

I would suggest the use of some additives such as glycerol, DMSO, betaine etc.

There are also commercial PCR additives, such as by Promega and Invitrogen.

Good luck.

[shivu]

Thanks a lot Mahmoud, I have not yet tried ready PCR master mix till now. I will try to do that. Hope that will be helpful.
Thanks a lot for the help.I

Originally Posted by m37786

dear shivu
can you try with ready PCR master mix such as Promega M7122
mahmoud manama

[shivu]

Originally Posted by jonoave

I would suggest the use of some additives such as glycerol, DMSO, betaine etc.

There are also commercial PCR additives, such as by Promega and Invitrogen.

Good luck.

Dear Jonoave,

Thanks a lot for the help.
Can you please provide me some protocol on how to use these PCR additives, I mean the amount !!
Any help will be highly appreciable.
Thanks

[shivu]

I was just wondering if someone else also has tried reducing the extension temperature during the cycle. If someone can please provide a protocol for the same, i m confused whether extension temperature during the cycle should only be decreased or the final extension too.
Sorry if you find my questions so naive, but i m new to this field, but slowly i will catch up !!

[jonoave]

Originally Posted by shivu

Dear Jonoave,

Thanks a lot for the help.
Can you please provide me some protocol on how to use these PCR additives, I mean the amount !!
Any help will be highly appreciable.
Thanks

I have used a mixture of DMSO, glycerol and dimethylformamide. I use bout 2-5% of the total reaction volume, of a regular PCR reaction setup. E.g. in a 50 ul reaction, I add 2.5 ul of additives.

You can try using google scholar and look up papers on PCR additives for more information.

[shivu]

Dear Jonoave,

Thanks a lot for the reply, i will try using what u have suggested. The good news is, i m able to amplify one out of three genes. I will try for other two also. Thanks a lot to all for their help.
Regards

[Icymoon]

I'm trying to PCR an AT rich terminator. I've tried using DMSO and failed. I really need the right protocol too