I am currently setting up the protocol for bisulfite sequencing from scratch in our lab, and I have a million questions (some maybe very silly..) but I am quite confused at the moment! I hope someone can help.
Up until bisulfite conversion I understand - extract genomic DNA and use appropriate kit for conversion (we have chosen Zymmo Research kit), with regular check-ups on quality/integrity, as well as use a control DNA.
But here the doubts start... we are going to use a 3130 genetic analyzer from Applied Biosciences as our sequencer. Now, although I have read and practised and tried all the primer design pages for bisulfite sequencing, I am confused as to what primers and PCRs to be carried out (or if just the One!).
Following my bisulfite conversion, is it possible to use the product obtained in a PCR with an appropriate polymerase/mix (we were thinking BigDye Terminator v3.1 Cycle Sequencing kit, Applied) and primers, and the end-product of this PCR use it directly for sequencing (after purification)?
Regarding my primers: I understand that the primers I get from places like Methprimer or Zymmo Research page are specific for bisuflite PCR, but since I have read so much about biotinylated primers, methylated specific, non-specific, etc. I am very confused as to which ones use and how many PCRs!
Any help or suggestions will be appreciated!!!