Dear researcher,
I am extracting DNA from IV plants which contain a lot of phenolic compounds and secondary metabolites, due to which the DNA extracted always contains impurities (I tried several ways to remove them).
Please tell me the ways to either purify the DNA or such PCR conditions/programs which could amplify the DNA.
I'm not sure the methods you have used for DNA purification, elaborating a bit would help us in finding a solution. I assume you're using phenol chloroform extractions? I can advise you to be very careful in not disturbing the aqueous-phenol interface when removing the aqueous phase. Don't be greedy, it's okay to leave some of the aqueous phase behind. If that doesn't work you can try commercial DNA purification columns, but I would advise trying the phenol chloroform extraction again as the DNA purification kits can be expensive. Even if the plant has lots of phenols they would be trapped in the organic phase and with proper centrifugation should not massively contaminate the aqueous phase. The problem with PCR is that your phenol contaminants can inhibit the reaction, so having pure DNA is essential for PCR.
Other researchers will comment on purification methods but a couple of things help pcr. First use enzymes that amplify out of whole blood ( These are often modified and run in buffers minimising inhibitors ). Remember the plant inhibitors often look like dna to UV light so you have less dna than you think. This is good as using much less dna ( therefore less inhibitor) often amplifies better. Inhibitors often chelate Mg ions which are essential to polymerases so along with less dna and more cycles I would try increased Mg ion concentration. try doubling Mg and use 1/4 or 1/8th amounts of dna to start or better titrate each in serial dilutions
Try diluting your template. Even if contaminants co-precipitate along with DNA, Less DNA=Less inhibitors. Try different dilutions: 1:10, 1:100, or even 1:1000.
Dear Sean Seltzer,
You guessed it right, I used phenol:chloroform:iso-amylalcohol for DNA extraction with CTAB.
I have used 3 different commercial kits but still DNA is not pure.
The pellet formed is very blackish brownish still.
Dear Paul Rutland ,
Thank you very much for your answer.
I did the same i.e made dilutions 1:1, 1:4 and 1:100 but I didn't increase Mg++ Concentration which I will do now.
Kind Regards
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