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Bisulfite genomic sequencing PCR


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7 replies to this topic

#1 killifish

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Posted 09 June 2009 - 06:57 AM

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

#2 epigenetics

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Posted 09 June 2009 - 08:41 AM

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.

#3 methylnick

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Posted 09 June 2009 - 01:48 PM

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

All comments and communication are my own personal ones, and are not tied to any of my affiliations. 


#4 killifish

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Posted 09 June 2009 - 01:56 PM

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.

#5 methylnick

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Posted 09 June 2009 - 02:01 PM

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

All comments and communication are my own personal ones, and are not tied to any of my affiliations. 


#6 killifish

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Posted 09 June 2009 - 02:55 PM

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?

#7 MoB

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Posted 09 June 2009 - 10:32 PM

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

#8 Michaelro

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Posted 19 June 2009 - 12:17 PM

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




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