When we try to purify phage DNA, we used a host bacteria, how do we differentiate that corresponds to the phage DNA itself and not the bacteria? Which molecular method could be used because i do not even know the phages that we have isolated?
hi,
The best method to isolate phage DNA could be the use of buffered phenol-chloroform extraction method.I am following this technique for the past one year and its working for me.There is a book "Current protocols in molecular biology"(vol1) to see the complete protocol,even though they are referring that method for isolating Lambda phage DNA you can optimize it accordingly .when you run the gel you can see the band above 10kb.To confirm this you can do a 16s rRNA specific PCR ,use host DNA as a control,you can see amplication(1.5kb) in the host DNA but not in the phage DNA which will confirm the result.To make sure that it is not the plasmid DNA use DNase and incubate before adding buffered phenol,bcz buffered phenol will cleave the protein coat of your phage and release its DNA so if you use DNases after that it will cleave your phage DNA.
Hope this will help you.All the best..
you avoid contamination by treating the lisates with DNAse before phage precipitation. The 16s DNA control proposed by Amrita is a good option to be sure that is not contaminated.
In addition to Susana's suggestion, you can also purify your phage by centrifugation on a CsCl density gradient. Don't forget to dialyze away the CsCl before proceeding to the phenol-chloroform extraction
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