I'm having a problem extracting DNA from phage colony for sequencing.
Currently we are using the standard protocol in the manual and getting very low yield or proteins. Any suggestion?
Dear Guanfan Mao,
You are requested to please provide the details of procedure you are following, so that we can answer you better.
thanks for your answer
the procedure we are doing is very simple as describe in the manual of the phage kit.
incubation of the phage with e.colli for 4.5h 9one colony in 1ml of e.colli over night growen) then taking 500micol adding PEG for 20min centrifugation then edding to the pellet iodid ethanol and centrifuge again
getting mostly protein
ant suggestion?
Another way to get around the problem is that do a quick PCR to amplify your insert and sequence the insert. (Just a touch of phage stock or 0.5ul will work for PCR). If you need to do many clones this could be the most efficient way to sequence all. Proper host cell lysis is key for a good phage preparation.
What phage are we speaking about? The majority of the phages will not grow if you put the plaque into 1ml of the stationary overnight E.coli. Try to put the phage to the activly growing culture ca. OD 0.4. If the phage is filamentous let it stand after inoculation about 1h without agitation and then grow overnight at 30C instead of 37C
i folloed phenol /chlorofome phage DNA extraction methhod for my vibrio phage and i got good yeild of DNA with streak, i need sugestion for avoiding streaking
https://www.researchgate.net/post/How_do_you_identify_if_the_isolated_bacteriophage_genomic_DNA_is_pure_only_from_phage?_tpcectx=qa_overview&_trid=53b66e77d039b1fb558b4587_2
Hi, could you please refer me to the specific method protocol you are using? (Specifically I would like to know whether you use NaI and if so, what molar concentration?). Thanks,
see link below.
http://www.openwetware.org/wiki/Gill:Phage_genomic_DNA_extraction#Reagents_required
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