plant genomic DNA isolation and PCR - does CTAB affect PCR ? (Jul/04/2007 )
i am using CTAB method for isolating genomic DNA from a variety of plants.
i use chloroform : isoamyalchol purification
washing with 70% alcohol
is there anything else to do ?
after gel checking the bands are clear , some are with RNA and protein ,,
when i did PCR using the good samples ,, the amplified band is too faint or none at all
does CTAB extraction affect PCR reactions?
if so how can i over come it ?/
any help appreciated
thanks in advance
I would recommend doing phenol:chlorofom:isoamyl (25:24:1) on top of CTAB, probaby do the PCI twice too. The most common PCR inhibitors from plant extractions are polysaccharides and polyphenolics. Any species that has a lot of sap or gum will have a lot of these so there is potential for inhibition there, check your "DNA" pellets, if they are brown or large, then you have some co-precipitated polysaccharides or polyphenolics. Unfortunately they are hard to get rid of. I can recommend the Qiagen Plant DNA extraction kits, they are quick and very good but not cheap.
Oh yeah, CTAB will affect your PCR too, make sure you wash the pellets well it should get rid of CTAB.
i will try to get a kit
and also try purification again ,
I use a modified CTAB method. It's long and labour intensive, but it's the method that gives us the best results in terms of DNA quantity and purity.
If you take confidence with the method and do all the steps properly, it won't affect your results as much as you have experienced.
I've never tried to use kits. Anyway, if they allow you to spare some time with a reasonable efficience, I will suggest you to make a try...
thanks very much please do send me the details , i will try it .
i am at my wits end , i tried phenol chloroform extraction