The problem is that in the gel the DNA bands are formed very very low. It is like fragmented DNA. I thought that the problem may be the precipitation of these phages with PEG 10% because another sample without PEG run normally on the gel.
I upload also the pic (sorry for the bad quality) in order to get a picture of the situation... The positions of interest are 2 to 8...
Hi!
Could you please describe your protocol in details? I'm not sure, that PEG is the problem, it will be very strange...
Regards,
Evgeny
Hello! Actually it is just a common Phenol-Cloroform protocol. It is described in Higuera G., Bastías R., Tsertsvadze G., Romero J., Espejo R.T. Recently discovered Vibrio anguillarum phages can protect against experimentally induced vibriosis in Atlantic salmon, Salmo salar. Aquaculture. 2013. 392-395: p128-133.
Yesterday I changed the density of the gel to 0,4% and I ran it for 3 hours at very low speed and I got really better results. You can check in the pic.
However, there is still this unwanted smear at the downside of the gel... This will definitily interfere with my results in the restriction analysis that I want to make. Do you have any ideas how to remove these fragments???
Actually, I have already done DNAase and RNAase treatment to the smaple and I am waiting for the results. But if the DNAase treatment works, on the one hand I will get rid of the unwanted smear but on the other hand it will denaturate also my phages DNA!... So, I am not sure about the next step. If you have any ideas please send me!
Thank you,
Panos
Dear Panos
As our team experience about PEG concentration don't have any interference with DNA extraction. We concentrated our phages by PEG 20% (8000) at 12000 rpm and we recruited Norgene DNA extraction kit. Consequently, there is good band of DNA and also we done RAPD-PCR for our phages. So I think it is referred to your extraction process not PEG.
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