Bisulfite direct sequencing + cloning - (Dec/10/2007 )
I have failed in before direct sequencing from the PCR porduct, so i tagged my primer with Sp6 and T7. Then i get a single band from my PCR product, then i cut the gel and purify it. However, i cannot perform direct sequence from the PCR product using Sp6 (forward) and T7 (reverse).
Seq. buffer 1.75 ul
Forward primer 1ul or Reverse primer 1ul
3.1 BD 1.5ul
dd H2O 2.75ul
96 'C 1 minute
96 'C 10 seconds
50 'C 5 seconds (25 cycles)
60 'C 4 minutes
4 'C hold
What is the key for direct sequencing?
Also, i have clone the PCR product into a plasmid. However, from same DNA sample. After cloning, show very different result. One show all 7 CpG methylated and remains C , the other show all converted to T.
Why is such result obtained?
Sequencing can fail for any number of reasons. Have you had much experience with sequencing? If you have strong product after the PCR clean up you are in a good position. Was this the case? If so then you probably need to fiddle with the volumes of BigDye and template you put into the sequencing reaction as they can cause big variation in the success of the sequencing result. How big is your fragment?
I got strong band in PCR but not very clear one. Also my fragment size is about 200bps including sp6 and t7.