MSP- not getting ANY products - (Feb/13/2013 )

Hi,
I've been trying to do MSP for months now and just can't get any products from my bisulphite treated genomic DNA.
I've been trying to amplify a region of the HIF1 promoter to repeat some work done in this paper:
http://www.nature.co...nc2010481a.html
But there is no protocol given in the paper and the reverse primer they say they use, i can't find anywhere in the target sequence.

I designed my own M and U primers using methprimer but cannot get products from my bisulphite treated genomic human DNA template with either primers. Tried another set of primers that i designed by eye but no luck either. (target DNA sequence and primer sequences attached).
I've tried Gotaq polymerase, Q5 hot start polymerase and KOD hot start polymerase. I've tried a range of annealing temperatures (40-65C), primer concentrations: 0.2-0.4 uM and input template amounts: 100-300 ng.

To add complication, i was getting amplification of the region 100bp upstream from this using primers designed for bisulphote sequencing, sowing my template isn't the problem.

I'm at a total loss, can anyone help?

Thanks

Ish

-ishmistry-

Your primers and sequence were not attached. You will never succeed with Q5 or KOD, which will not amplify the u's in bisulfite modified DNA. Use Taq.

-phage434-

Thanks for your reply!
Does the enzyme need to be HotStart?
I think I've successfully added the attachment now.

-ishmistry-

yes, hotstart taq is very helpful.

You may want to design a set of universal primers outside the MSP amplicon region to fist amplify just the bisulfite modified DNA, and then use the PCR product as template for MSP.

BTW, there is a mistake in your right U primer picked by eye: AAATGAATAAAAATGAAAT

-pcrman-

ishmistry on Wed Feb 13 18:25:28 2013 said:

Thanks for your reply!
Does the enzyme need to be HotStart?
I think I've successfully added the attachment now.

As I mentioned in some posts, you should try the PfuTurbo Cx HotStart polymerase (Agilent Technologies), it really helped me! This is the only one in the market (as far as I know) that is proofreading and also reads uracil.

-vilperte-

pcrman on Wed Feb 13 20:44:31 2013 said:

Oops, the primer mistake was from me typing it into here wrong. The universal primers sound like a good idea, do you have any advice about how I would go about picking which to use? And where to get them from? Would standard primers work or would they have to be specific to bisulphite treated DNA?

Thanks for your help

Ishna

-ishmistry-

Basically universal primers are the same as bisulfite sequencing PCR (BSP) primers and are specific for bisulfite converted DNA. You can either pick manually or use the MethPrimer program by choosing BSP primer design.

-pcrman-

great thanks, i'll try that

-ishmistry-