effect of ethanol and dna concentration in pcr - (Aug/13/2005 )
hi everyone again.
I just want to ask what is the effect of a few ethanol on dna sample in pcr? coz i rehydrate my isolated dna in tris-hcl even if there are still traces of ethanol in it, which is about 5 ul i think, coz i heard that it would even be bad if i leave the sample overnight and the dna get so dehydrated.
one thing more, i would like to ask , i rehydrated my dna sample in 100 ul trsi-hcl. In pcr, do you usually make dilutions of your isolated dna or you take 1 ul directly from the original tube? My samples have varying dna concentrations ( some bands visible in agarose gel, some are not, some range from 30 ng, some about 1-3 ug) I am afraid that it will affect my pcr products. I only need around 10 ng ( based on journal) Can you give me some advise concerning this? thank you.
If your DNA takes longer than 15 min to dry at RT then you have left too much EtOH behind. Spin your samples again an suck off the remaining ETOH.
Better DNA sequencing results
Qiagen reports that 5% ethanol causes "significant inhibition" of PCR reactions. (D. Loffert, S. Stump, N. Schaffrath, M. Berkenkopf and J. Kang, 1997 PCR: effects of template quality. Qiagen News 1: 8-10.)
Wow no wonder I couldnt get any band for PCR if I'm in a hurry to dry the DNA from EtOH hehe
Patience is virtue haha