DNA purification - (Oct/11/2004 )
I'm new to this forum. thanks for this great resource.
I'm working on dna methylation and after repeated trials my pcr doesnt work. I am using chemicon kit. I am using the dna extracted from the fresh tissue samples.
the wild type unmodified dna samples work good but the modified dna with the methylation specific primers doesnt work.
one thing I noticed is the purity ratios on the spectrometer reading are shown as 20 and sometimes 30. its wiered.
can anyone suggest the best purification method and is it better to purify before or after midification.
appreciate ur help.
You said you are using Chemicon kit, that is the CpGenome DNA mod kit, right? There are purification reagents coming with the kit. After you finish the protocol, your DNA is purified and ready for PCR with good recovery.
What is purity ratios, 280/260? What solution did you use to resuspend your DNA at the last step of modification?
thank you pcrman
yes ur right .iam using the cpGenome modification kit.iam resuspending the dna in TE buffer as recomended. 25 ul for 1ug of starting dna. the purity ratio iam talking about is 260/280.I think there must be something wrong with the machine .
what is the yield you usually get when u use 1ug of DNA.
after talking to the tech support he suggested using reagent 4 .so iam trying that today.let u know of the results soon.
You didn't use reagent IV? You should have used it which is some type of carrier probably glycogen.
We don't measure the purity or concentration after modification because it is unnecessary. According to the manual of the kit, 1 ug of starting DNA will yield 40 ng/L modified DNA that is enough for 20 PCRs (use 2 ul of the modified DNA in a 20 ul of PCR reaction).