Hi all. I am trying to collect high numbers of intact phages from bacterial cultures while avoiding contaminating DNA from the lysed bacteria; this is for eventual PacBio sequencing of the phage genomes. So far I am working with the Ambion/Invitrogen Turbo DNAse kit for removing DNA from RNA preps. My issue is that I have so far been unsuccessful in completely eliminating a 16S PCR signal (it is weakened) by DNAse treatment while maintaining phage integrity. Using elevated levels of DNAse, a 37C incubation temperature, and extended incubation times (two 45-minute periods with fresh DNAse added), I still get a weak 16S and up to 80% loss of intact phages. Do any of you have experience in successfully accomplishing this sort of procedure? Suggestions and types of wisdom will be much appreciated. Thanks!
First, full disclosure: I have some experience with phage, but not sequencing phage with PacBio. We used to purify the phage after DNAse treatment. There are 2 methods that we used (depending on what we were going to do with the phage). 1) precipitation of the phage using PEG and NaCl. 2) pelleting the phage using high speed centrifugation followed by slow resuspension. After these procedures, most of the phage were intact as determined by titer. You could do multiple rounds of DNAse treatment followed by purification to reduce the 16S signal. But remember PCR is really sensitive, so you may never completely get rid of it. Some DNA may be adsorbed to the outside of the phage coats. On the other hand, why get rid of it at all? You know its sequence, so just remove those sequences from your data before analysis. Practically all sequence analysis software has methods for getting rid of contaminating vector DNA, so just treat the 16S or other bacterial host DNA as contaminating DNA.
Best of luck.
Jeff,
You could try column chromatography to initially bind the protein coat of the phage, or conversely bind the free DNA while eluting the phage. Being purely speculative perhaps trying to drive the DNA using electrophoresis into agarose would leave behind the phage in the well - totally not sure of the charge of the phage you are studying so this is speculative.
Suggest using CsCl equilibrium density gradient centrifugation. The phage band should have no free DNA contamination. See attached for methods.
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