Bisulfite modification kits and PCR
Posted 19 July 2004 - 03:56 PM
We are trying to amplify a PCR product of about 400 bp for cloning and we also need to use the template for MSP.
Has anyone found that DNA is more easily amplified from one of these kits compared to the other?
Thanks for your help!
Posted 19 July 2004 - 05:31 PM
If your DNA is from cell line or fresh tissue, the chances of getting 400 bp amplification are high and the modified DNA should be also OK for MSP.
For modification, use 1-2 ug DNA and elute in 30 ul water at the last step. Use 2 ul for a 20 ul PCR reaction.
If you are amplifying DNA for sequencing or cloning, start at 40-45 cycles and hot start your PCR or better use some hotstart taq such as the JumpStart from Sigma which really makes a big difference.
Posted 20 July 2004 - 12:19 AM
I think it is important to have clean DNA otherwise it may inhibit the PCR later. Do you rountinely Phenol/Chloroform extract? It helps a lot.
Posted 20 July 2004 - 08:00 AM
At what stage you do this, before or after modification?
Do you rountinely Phenol/Chloroform extract? It helps a lot.
OK, I got it from your another post. You mean before modification. Do you think that is crucial, since you have another chance to purify your DNA after modification?
Edited by pcrman, 20 July 2004 - 08:07 AM.
Posted 20 July 2004 - 06:27 PM
Posted 10 August 2004 - 09:26 PM
It seems true. Recently I modified two batches of DNA at the same time with one was not very clean (recoved from luciferase assay). The result was the "dirty" DNA was not completely modified as shown by sequencing.
start with really clean DNA