Amplify long fragment from bisulfite treated template - for bisulfite sequencing (Jan/27/2005 )
hmmmmm
I have done a fair whack of BSP reactions with no problem. I was wondering if your comment is true, have you tried a PCR at the normal temperature of 72C for the extension step?
I just tried re-amplifying my samples with the same primers I used for the initial PCR. I did it with the following PCR cycling conditions:
95 15 min (Hot start for the enzyme)
40 cycles of <
95 30 s
53 30 s
gradient of 53 to 72 for 1 minute >
Melting curve 60 - 75 degrees
There was no amplification of the product for extension temperatures at 68 or above. Optimal (shortest number of cycles) amplification occurred at about 61 degrees. Much poorer amplification occurred below 60 and above 64.
These samples are an extreme case -- bisulfite conversions of bacterial DNA with a 26%
GC content to begin with, yielding a 13% average GC content following bisulfite conversion.
That's really interesting and informative phage, thanks!!
If I have trouble with my PCR's I will give your protocol a go!
Nick
I had the same problem with long fragment PCR with bisulfite treated DNA. The very first time I got the long product but afterwards, no PCR product at all. I tried the same DNA template with another set of primers which amplify a 300 bp product and that was successful.
I doubt if this is because bisulfite treated DNA is more fragile so that I failed to amplify the long fragment.
By the way, how long can you keep you bisulfite treated DNA before the PCR?
I have successfully amplified bisulfite treated DNA which has been stored at -20C for almost two years.
[quote name='pcrman' date='Aug 25 2005, 03:10 AM' post='22943']
[quote=wujinfang2004,Aug 23 2005, 09:35 AM]By the way, how long can you keep you bisulfite treated DNA before the PCR?
[/quote]
I have successfully amplified bisulfite treated DNA which has been stored at -20C for almost two years
strong!!!!!!!!! can i use taq polymerase in hot start pcr?
strong!!!!!!!!! can i use taq polymerase in hot start pcr?
Sure can :-)
Hi,actually my PCR works with the extension at 72C.But my problem is the tmeplate can't be sequenced out, I am wondering if the template now is CG lowand with a long run of TG after bisulfite..does anyone encounter the same problem?Thanks.
Standard sequencing reactions run the extension phase at 50C, which means they are immune to this problem, at least in my experience.
but my extension temperature is 60C instead of 50C in sequencing reaction...thinking of denature the reaction mix at 98C for 5min cos afraid there is CG rich content in the template..will it work?will try and see how..