I was wondering if anyone could provide an opinion on the difference in PCR amplification effiency from DNA that has been Bisulphite modified using either the MethylEasy Kit or the EZ DNA methylation kit. 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! -Kiwicloner- -------------------------------------------------------------------------------- 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 μg DNA and elute in 30 μl water at the last step. Use 2 μl for a 20 μl 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. Good luck. -pcrman- -------------------------------------------------------------------------------- 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. -methylman- -------------------------------------------------------------------------------- QUOTE Do you rountinely Phenol/Chloroform extract? It helps a lot. At what stage you do this, before or after modification? Edit: 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? -pcrman- -------------------------------------------------------------------------------- -methylman- -------------------------------------------------------------------------------- start with really clean DNA 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. -pcrman- -------------------------------------------------------------------------------- |
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