I need pure phage DNA of my T4 phage for preparing standards for qPCR. I tried 3 different protocols (precipitation with PEG/NaCl, with/without DNase treatment) to prepare T4 phage for DNA extraction (used Norgen phage isolation Kit). After DNA extraction I did a standard PCR to check if there is any E. coli DNA left. From all products I also made agarose gels. Maybe someone can help me to interpret my gels after DNA extraction. I have relative clear bands of my phage DNA, but fat smear at 200-300 bp.
I. without DNase treatment: bright bands for E. coli DNA after PCR
II. with DNase treatment: really weak bands for E. coli DNA after PCR III. with DNase treatment 2nd protocol:
one sample: weak bands for E. coli DNA after PCR
second sample no bands for E. coli DNA after PCR
Is the smear at 200-500 bp really E. coli or could it also be some phage fragments?
thanks a lot
to remove these smear please treat your lysate with RNase A alongwith DNase treatment, secondly for inactivation of DNase use a little higher temperature say 5 degree more than mentioned in the literature. Use DNase/RNase free water for elution.
Thanks for your answers, I should have mentioned: first I centrifuged the lysate 2 hours (8000 rpm) and then filtered the supernatant at 0.45 and 0.22 μm before PEG/NaCl/DNase.
Inactivation of DNase I did for 10 min at 75°C. Why should I add RNaseA? I did a qPCR with the 2nd sample (2nd protocol with DNase) and it worked fine. I still have no idea what this smear could be.
Thanks for all your answers. I tried the treatment with the RNase and it worked. I have no smear and fine bands for my phage at the gels now.
Thanks Janin Dineman. infact i had worked continuosly for 7 months and resolved this isuue after 7 months.
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