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Bisulfite genomic sequencing PCR - PCR conditions (Jun/09/2009 )

Hello,

Can anyone help me with the BS-PCR?

I have designed primers for BS-PCR using Methprimer and I cannot see any bands with my primers. My bisulfite conversion worked well as I see bands with another set of primers.

One thing I notice is that the primers have low GC content (20-30%). My PCR condtions are 95 for 10 min, 40 cycles of 94 for 30 sec, annealing temp. for 30 sec and 72 for 30 sec and final extension at 72 for 8 min.

I really appreciate any help.

Thank you

-killifish-

Hi,

Just wanted to share my experience.

I did the same, used Meth primer for BS-PCR.
For Fibronectin gene, ordered two sets of primer. 1st set did not work. 2nd did. Also it took good amount of time to select right A temp. I had to increase dNTP also. Try different MgCl2 conc also.
Besides these,
I think, you might first lower the A temp. If you find out atleast some thing then, you can generate 1 more primer for same CpG. after doing first round of PCR, you can use the 3 rd one along with either one from 1st two to do 2 nd round of PCR. It will help for sure.

lets see what experts say.

-epigenetics-

Very good suggestions Epigenetics.

A couple of things to add, you could try a temperature gradient to optimise the Tm of your primer set. I have never liked methprimer as a design program and tend to design by eye. There are the parameters I use pinned in this forum.
PCR and primer tips


Another thing you could try is to design more primers such that you have a nested PCR strategy and perform two rounds of PCR on your samples.

good luck

Nick

-methylnick-

Thanks to both of you for the suggestions. I did try the gradient PCR and didn't find a single band. Now I am trying with much lower range to rule out the annealing temperature as the issue. I will try few other sets of primers.

Thanks once again.

-killifish-

I have added the link in to my post above! good luck! :lol:

-methylnick-

Thanks, Nick. I really appreciate it.

I have one more question. I have never seen anyone mentioning in papers or protocols about quantifying the concentration of bisulfite converted DNA. Why isn't it important? I tried to measure it and multiplied the absorbance with 40, as it is single stranded DNA. Is it correct or should I use 33 as used for single stranded DNA. I noticed that my A260/280 ratios are between 1.4-1.5. Any thoughts?

-killifish-

I agree to Veteran. Spend more time in designing primers and you'll save time and money during optimization. I also never trust a design-program but always rely on my own abilities. I use NetPrimer for calculation of annealing temp and testing for dimers, hairpins, cross-dimers etc... I design all primers to an annealing temp of 55C, all primers are in between 20 - 25 bp and cover 3 - 5 conversion-sites. Design short PCRs as the template DNA is highly fragmented after bisulfite treatment. PCRs from 120 to 200 bp are easy to realize even with a small amount of template DNA (1-10 ng). For PCRs of > 300 bp the amount of template DNA must be increased.

I use standard Qiagen HotStart Taq (1 unit per PCR), 2.5 mM MgCl2 and the only variable are primer-concentrations. I run 40 cyclesand receive good results for most of my PCRs without any optimizing effort.

Good Luck..

MoB

-MoB-

killifish on Jun 9 2009, 05:57 PM said:

Hello,

Can anyone help me with the BS-PCR?

I have designed primers for BS-PCR using Methprimer and I cannot see any bands with my primers. My bisulfite conversion worked well as I see bands with another set of primers.

One thing I notice is that the primers have low GC content (20-30%). My PCR condtions are 95 for 10 min, 40 cycles of 94 for 30 sec, annealing temp. for 30 sec and 72 for 30 sec and final extension at 72 for 8 min.

I really appreciate any help.

Thank you


Try low elongation temperature
64C works good for me
Low elongation temperatures 64-66C are recommended for bisulfite PCR
Good Luck

-Michaelro-