I want to do methylation specific PCR. So, I am doing bisulfite treatment. In some protocols 3M stock of sodium bisulfite is used which finally becomes 2.6 M in solution (Herman et al). some uses sodium metabisulphite in final conc. of 2.31 M. I am using 1.5 M stock of sodium metabisulphite . As 1 mole of sodium metabisulphite is equal to 2 moles of sodium bisulphite . thus this will be equivalent to 3 moles of sodium bisulphite and the final conc. of sodium metabisulphite is 1.3M with this stock. so, according to me it should be equivalent 2.6 M of sodium bisulfite as is there in Herman's protocol? Am I right or I should treat sodium metabisulphite as sod. bisulphite and use the same conc. as mentioned for sod. bisulphite. Does everybody uses this sod. metabis. for bisulfite treatment and use at same conc. as is mentioned in the protocols for sodium bisulfite sodium bisulfite is also used? I am really confused with this sodium metabolism. and sod. bisulphite. So, please help me to overcome this confusion.
2.6M is sodium bisulfite.metasulfite. Infact it is saturated bisulfite solution and hence 2.6 or 3M does not make a big difference. I read somewhere 9M (there is a trick in making such bisulfite solutions) used for DNA methylation studies.
Try kits from:
http://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion/ez-dna-methylation-kit
Otherwise read the following protocol:
http://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion/ez-dna-methylation-kit
Good luck
I couldn't find the concentration, but I used the kit linked below, and it worked great. I did probably close to 100 conversions and it was very consistent and reliable every time. Maybe email Qiagen and try to get the concentration out of them, and mimic that.
http://www.qiagen.com/us/products/catalog/assay-technologies/epigenetics/epitect-plus-bisulfite-kits/
In short: you are right to use half the amount of sodium metabisulfite. So, if you want a 2M concentration of sodium bisulfite, prepare a 1 M solution of sodium metabisulfite.
I have added a link below from a book so that you have a reference to back this up :) (I am not sure why this forum added all the grey text, but if you click on the link it will take you to page 79 of the book)
Funnily enough the paragraph starts like this: "There has been some confusion about concentrations of sodium bisulfite solutions." :-D
http://books.google.de/books?id=_yv4JklV4OoC&lpg=PA79&ots=WfqTwhB6cv&dq=bisulfite%20conversion%20protocol%20metabisulfite&hl=de&pg=PA79#v=onepage&q=bisulfite%20conversion%20protocol%20metabisulfite&f=false
Hi Lalita,
I agreed with previous replies that 2.6 M or 3M concentration doesn't make big difference. However, I could suggest to perform a control pilot MSP experiment using proper control for bisulphite treatment like performing b-actin MSP. Further, we also always perform DAPK MSP for Leucocyte (negative); in-vitromethylated Lucocyte (positive control) and whole genome amplified Leucocyte (negative control). If that works after your bisulphite treatment then you can proceed easily for other genes.
Following are the sequences:
b-actin MSP Frw: 5' TAGGGAGTATATAGGTTGGGGAAGTT 3'
b-actin MSP Rev: AACACACAATAACAAACACAAATTCAC
DAPK Meth MSP Frw: GGATAGTCGGATCGAGTTAACGTC
DAPK Meth MSP Rev: CCCTCCCAAACGCCGA
DAPK Unmeth MSP Frw: GGAGGATAGTTGGATTGAGTTAATGTT
DAPK Unmeth MSP Rev: CCCTCCCAAACACCAACC
However, I have to admit that we also used bisulphite kit from Zymo research and its pretty consistent as I have performed this on >1000 of patient samples so far.
http://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion/ez-dna-methylation-kit
All the best and let me know if you need further help.
regards,
Tushar
Yes, indeed. We used to use our own protocol for bisulfite conversion, but we have switched to kits now, mainly becuase we get more stable conversion rates (very rarely under 97% with kits). The Zymo kit that Tushar recommends is what we use too.
You might be better using the MethyEasy Eceed Kit from Human Genetic Signatures. This one has been used for whole genome sequencing and has proven to be reliable even using small quantities of starting DNA.
Commercially available sodium bisulfite powder is a mixture of sodium bisulfite and metabisulfite of unknown proportion. Consequently, the final concentration of HSO3- can only be estimated. Since the sulfonation reaction (step 1 of the bisulfite procedure) is in equilibrium, you should use a maximum concentration of HSO3- to push the equilibrium to the right (in favor of cytosinesulphonate). I would recommend to use bisulfite/metabisulfite as saturated solution (5.2-5.7 M HSO3-). You can try our protocol (http://methdb.univ-perp.fr/cgrunau/methods/bisulphite_engl_mc.html) but I would also trust the kit recommended by Douglas Millar since it was he who contributed largely to invent the original bisulfite method.
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