It looks like people who are sequencing clones after bisulfite PCR + cloning always prepare plasmid minipreps for sequencing. I have tried a much faster way to do : just do a PCR with vector primers (M13 Forward and Reverse) from a single colony (just pick a colony with a pipet tip, stir the tip in 10 microl H20 and use 1 microl for PCR). Then, after PCR, you just need to purify the PCR product and sequence it.
Does this sound silly to anyone ?
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Posted 08 October 2009 - 06:35 AM
Hi,
not silly at all, you certainly don't need a miniprep. I guess people often don't do colony pcrs at all - in my previous lab i always did minipreps and restriction digestion instead of colony pcr, stupid, but it was a rule. Second reason could be that they try to avoid potential mutations that could be introduced by second pcr reaction, but the fragments amplified are usually so short that it seems improbable.
BTW, sequencing certainly goes much better on amplicons than on plasmids
g.
not silly at all, you certainly don't need a miniprep. I guess people often don't do colony pcrs at all - in my previous lab i always did minipreps and restriction digestion instead of colony pcr, stupid, but it was a rule. Second reason could be that they try to avoid potential mutations that could be introduced by second pcr reaction, but the fragments amplified are usually so short that it seems improbable.
BTW, sequencing certainly goes much better on amplicons than on plasmids
g.
