MSP condition - (Feb/28/2006 )

Hi,

I am new in MSP analysis. I read several papers and found lots of researchers used the PCR condition described by Dr. Baylin in PNAS. 1996; 93: 9821-9826. Since the buffer they used is a little complicated, I wonder if regular PCR buffer is ok for MSP analysis. If anyone can provide a recipe of PCR reaction which works well in your system, I would be very appreciated. Thanks!

yft

run off the mill PCR buffer works fine for me.

With most PCR primers, the conditions may require optimising, Tm and Mg concentration are the two factors to get the assays working.

Nick

Hi, Nick,

Thank you very much for your reply. I am also reading the protocol of bisulfite modification you posted before. My question is:

1) Except Sodium bisulfite, I can make stock solution for NaOH and Hydroquinone. When I use it, I just need to dilute them, right?

2) I am a little confused about Sodium Bisulfite solution. We have product Sodium Metabisulfite from Sigma (S-9000). If I want to make 3M Sodium Bisulfite solution, should I just make 1.5M Sodium Metabisulfie based on molecular weight?

3)Is there any product from Qiagen can replace Promega Wizard DNA Clean-Up System?

4)Have you tried EZ DNA Methylation Kit? Do this kit convert completely?

5)I read several papers. Most of them use Bisulfite/PCR buffer. Can I just use regular PCR buffer? I am not very clear why specific buffer is required for MSP?

6) When you set up positive control by using CpG Methylase, how many units of methylase do you use to treat 1ug DNA? How long? Do you digest genomic DNA before you treat them?

Your help will help me lot. Thank you very much!

YFT

hi to answer your Q's,

I make NaOH and Hydroquinone fresh each time, Concentrated NaOH reacts with Air to give sodium carbonate...furthermore Hydroquinone solution is light-sensitive and goes brown sitting at RT.

2) Indeed it maybe a little confusing....you want 3M bisulfite ions and it's slightly different for bisulfite and metabisulfite, however I have tried a supersaturated solution of both (slightly above 3M) I knew it was supersaturated as there were salt grains that wouldn't dissolve on the bottom. The solution works fine for conversion.

3) I am sure there is a qiagen kit, the promega kit is essentially a desalting resin. So if you can find a qiagen equivalent (remove unreacted salts from solution but leaving the DNA behind) then I am sure it will suffice.

4)I have heard of this kit, on this forum, there seems to be many problems people post about it more than anything. Might I reccommend MethylEasy, it's aussie made and works very very well.

5) specific buffer is used to ensure optimum hyb conditions of the primers in a MSP reaction, normal PCR buffer will usually suffice.

6) SssI methylase is what is used to methylate CpG. There is usually a pack insert with the enzyme and this will tell you the specific activity of that enzyme.

good luck!

Nick

Nick,

Thank you very much. Your answer does help me lot. Thanks!

YFT