Bisulfite sequencing PCR worked - (Feb/08/2013 )
I've been reading a lot of topics here, trying to get as much information as I can.
After 1 month struggling with my BS PCR, I managed to amplify my fragments.
I've tried to convert my DNA again, I changed twice my polymerase, I tried gradient PCR, without any results.
What really helped was: decrease the extension temperature for 65C and increase the time for 2 minutes (even with fragments around 300bp). Increasing the cycle number can also help it.
So, thank you for all the people that has been postings tips and trying to help the others. It was really useful for me!
Now I can show some results for my supervisor!
Congratulations for your results . This forum is indeed very helpful. Whatever questions I have in my mind I just come here and ask....the good thing is we get quick reply if we ask the questions in a right way (everybody should be able to understand what we want to ask). Thanks to all members and of course to Bioforum to make this platform for us
Glad to hear that you finally got it to work. Can you post your detailed PCR conditions here so that your experience could be helpful to others?
Of course I can!
The final concentration of the reagents:
0,2mM of each dNTP
0,5uM of each primer (I'm still trying with less primer)
1U/20uL of PfuTurbo Cx HotStart DNA Polymerase
60ng of DNA (quantified in Nanodrop - using factor '33' for ssDNA)
1) 95C - 3 minutes
2) 95C - 30 seconds
3) 50C (annealing temp. of the primer) - 30 seconds
4) 65C - 2 minutes
5) Repeat steps 2, 3 and 4 - 40 times
6) 65C - 10 minutes
Good luck everyone
Just one more thing!
If you still don't see an amplicon after the PCR, try to run a 2nd PCR with the same set up, but with only 25 cycles.
That is nice. thanks for sharing. Phage434 explained why lowering extension temperature is important in another post as shown below.
phage434' date='11 February 2013 - 10:40 AM on Mon Feb 11 18:40:25 2013 said:
The EXTENSION temperature should be lower as well. Many high AT regions will not extend at 72. Try extending (for longer times (double)) at 63 or 65. You are using a Taq polymerase, correct? High fidelity polymerases will not read the uracils in bisulfite modified DNA. How are your primers designed? Are you certain they are correct (it is very easy to get confused!)
Post Source: No PCR Products after BSP