I have pure colonies of Enterococcus on plates and I have to extract DNA.
I've tried 10 microg/mL of lysis buffer, incubating for half an hour as in protocol- it didn't work. Then I tried boiling cells with lysis buffer - it didn't work.
any other methods?
I use E. Faecium ERI 1 and E. Faecalis ERI 4 as controls. When I run agarose gel, it's completely empty, I can just see the ladder.
Is there lysozyme in your lysis buffer? I also had the problem with enterococci but with a lysozyme incubation step it worked.
First treat with lysozyme and then you can use Qiagen kit for DNA extraction. hope it will give good result.
Here I send you the protocol for DNA extraction so easy and hope that will be OK
I succeeded by suspending a colony in a 2% SDS lysis buffer and incubating it at 95°C for 2min. Then I used 1ul of this suspension in my PCR.
Otherwise you might try phenol/chloroform DNA extraction. For me that works for almost all bacteria. If you want I can send you a protocol (I don't have it here right now).
Agreed with Ibrahim , and you can prepare .07% agarose gel for the DNA run
Regards
Aamir
Wow, this was my first question here, and I've got great answers! I am very thankful :) i will probably try all of them.
lysozyme is in conc. 10 mikrograms/mL.
I will let you know what is the best.
You can please refer to the following links to get certain valuable details:-
http://www.ncbi.nlm.nih.gov/pubmed/11853234
http://www.scielo.br/scielo.php?pid=S0100-879X2000001100002&script=sci_arttext
https://books.google.co.in/books?id=2pZ5qmoxaIEC&pg=PA170&lpg=PA170&dq=extraction+of+DNA+from+Enterococcus+for+PCR&source=bl&ots=lnBOk_rh0g&sig=GUG9NFFOzdlQn6oI405YRVl2OhM&hl=en&sa=X&ved=0CE4Q6AEwBmoVChMIguDArbnixgIVEB6OCh0VMAST#v=onepage&q=extraction%20of%20DNA%20from%20Enterococcus%20for%20PCR&f=false
I am having the same problem as yourself using the same controls. did you find a solution that seems to work best for you?
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