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BSP PCR for help


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#1 Jacob2000

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Posted 21 September 2009 - 11:53 AM

Hi all,

This is my first post here. I would like to find a solution to my BSP problem. I designed several pairs of primers (MethPrimer) and used genomic DNA of liver cell line as gradient PCR template to calculate the best annealing temperature. Most of them were 45 c ~ 53 c. They all were pretty good under specified temperature (PCR product size is less than 300bp). The problem is, when I applied them to genomic DNA of liver tissue (QIAGEN blood and tissue kit) as well as fresh liver tissue, they failed to work (no any bands found on a gel). The problem occurs even when I quadrupled the DNA template amount- from 50ng to 200ng. For the bisulfate conversion process (Activemotif Meth Kit), I found that the yield of tissue DNA after bisulfate treatment was only one third or even less compared to cell culture DNA after the same treatment, e.g. recovery of tissue DNA with bisulfate was 400ng/2 μg and of cell 1200ng/2 μg. So what could I do? Shall I use a restriction enzyme, which does not digest the DNA within the region of interest, to digest tissue genomic DNA in order to increase bisulfate conversion efficiency? Reamplify the PCR product for 30 cycles? Try nested PCR? Or other suggestions for my troubleshooting? :)

PCR condition is following:
50 ng bisulfate isolated DNA
2 μm primer
0.5 μl dNTP (25 μm)
0.2 μl Taq (NEB)

95 c 5 min
35 cycles of
95 for 30 sec
Gradient (45~53 C) for 1 min
72 c for 3 min
72 c for 10 min before 4 c storage

So I am looking for any help in trying to get my MethPCR working. I would appreciate any help that your experts could provide.
Many thanks. :P
Jacob

#2 methylnick

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Posted 21 September 2009 - 01:46 PM

with the yields you are getting, you should get amplicons readily.

I think 2umol of primer is a bit much, do you get big primer dimers?

We routinely use 0.2umol in our PCR's try reducing the amount of primer.

I am not a big fan of methprimer either.

#3 Jacob2000

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Posted 23 September 2009 - 11:45 AM

Thanks methylnick. I will try it.
If it still not working, are there any other suggestions? Thanks again.

#4 methylnick

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Posted 24 September 2009 - 11:50 PM

I would suggest redesigning your primers with Methyl Primer Express.

Nick




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