After isolation of genomic DNA from E.coli using phenol/chloroform method, the sample shows RNA contamination.
(i) If the sample is treated with RNase enzyme is it necessary to remove the enzyme RNase to do PCR?
(ii) What suitable conditions are required for PCR using E.coli genomic DNA with primers having Tm of 59-60 degree celsius ?
Thank you
You could use a purification column (e. g., Macherey-Nagel Nucleospin) to remove RNase from your DNA sample.
Regarding your other questions: (i) no, it is not necessary to remove RNase to do PCR, (ii) you can use standard conditions, like 100 ng template DNA, 0.5 µM of each oligonucleotide, 0.2 mM dNTPs, etc. I would run a temperature gradient to identify the optimum annealing temperature of the oligonucleotides (e. g., 45, 50, 55°C).
Best regards.
You can repeat the phenol:CHCl3 extraction. Try not to get too close to the interphase, though.
And no, it is not necessary to remove RNAse to perform PCR. As a matter of fact, you do not need to remove RNA either.
Hope it helps!
Another round of Phenol/ Chloroform extraction will remove the RNAse.
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