DNA methylation - (Jun/04/2008 )

Could some one please point me a detailed protocol how to do bisulfite sequencing? I am kind of confused what PCR is done followed by sequening?
Thanks.

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Follow cloning protocol, do cloning for further sequencing.

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What I am doing is I have identified cpG island by bioinformatic approach and when I design primers for bisulfite sequencing it gives a product if ~ 150-200 bp Then when I have to sequence this PCR product:
1. Should I use same primers which I used to amplify Busufite amplify DNA?
2. The prduct size if ~150-200bp so in that situation (1 above) I will get sequence of only 150-200 bp?
Please clarify.
Tahnks.

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What I am doing is I have identified cpG island by bioinformatic approach and when I design primers for bisulfite sequencing it gives a product if ~ 150-200 bp Then when I have to sequence this PCR product:
1. Should I use same primers which I used to amplify Busufite amplify DNA?
2. The prduct size if ~150-200bp so in that situation (1 above) I will get sequence of only 150-200 bp?
Please clarify.
Tahnks.
[/quote]

You could use the same primer (either F or R) for sequencing directly with PCR products. However, the sequence you are given will be about 60- bp shorter than the PCR product size depends on your primer length. If you do cloning after PCR amplification, you can use different primer designed on the basis of plasmid, and get full sequence of PCR product.

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step 1. bisulfite convert your DNA at 50 deg overnight
step 2. clean up your dna
step 3. PCR your fragment of interest. Remember that BS conversion will lead to 2 separate strands - top and bottom, and you have to decide which to use as template. Either one will do, but depends on Tm's etc.
step 4. clean up PCR product
step 5. TA clone into plasmid
step 6. pick colonies, blue/white selection etc
step 7. mini prep clones
step 8. sequence either reverse or forward. Seq Primer can be also anywhere. it can be the same as the PCR primers or outside in the cloning region, lacZ' or T7, etc etc

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Thanks for the info One question in some protocols two step PCR is done while other paper suggest regular PCR using two degree less tm of lowest tm primer? any suggestion about that please.

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