I want to isolate the total DNA from bacterial cells. Does anyone know a good method?
Dear Shabeer Ahmad.
when i want to extract DNA from xanthomonas, I followed method described by Llop et al (1999). briefly as follow
1000 µl of the bacterial suspension (106CFU m-l) were transferred to 1.5 ml microcentrifuge tube and centrifuged at 10000 g for 5 min and supernatant were decanted. The pellet was resuspended in 500 micro liter extraction buffer (200mM Tris HCl pH 8, 250 mM NaCl, 25 mM EDTA, 0.5% Sodium DodecylSulphate, and 2% PolyvinylPyrrolidone). The tubes were shaken for 60 min at room temperature. Then the tubes was centrifuged at 5000 g for 5 min, and 450 micro liter of the supernatant mixed with 450 micro liter of isopropanol and left 30 min at room temperature. After centrifugation at 13000 g for 10 min, the supernatant was discarded, the pellet dried on air, and resuspended in 100 micro liter sterile water.
you may try it. Good luck
Hi Shabeer.
This is a somewhat old fashioned method but it gives good yield, is easy to perform and gives very clean DNA.
Use whatever method you prefer to lyse 1.5 ml overnight culture. Digest samples with proteinase K. Make your solution to 0.5 ml total volume and then do a normal phenol extraction. Add 1/10 volume of sodium acetate. Then add 0.6 volumes of isopropanol and mix gently. The DNA should start to precipitate and be visible as a white shadow. Spool the DNA onto a heat sealed glass Pasteur pipet. Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec. Resuspend DNA in 100-200 µl TE buffer.
Good luck
Jesper
The addition of Lysostaphin 0.2 mg/ ml in the pre- lysis step is very helpful ( incubation 60 min at 37 •C) before the addition of proteinase K.
http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Genomic%20DNA/UM_gDNATissue.pdf
See support protocol for Gram- positive bacteria.
You could combine it with Jespers suggestion.
All the best
Bea.
Ps:
Did anybody try this:( Lysozyme instead of Lysostaphin)
http://www.protocol-online.org/prot/Protocols/A-Simple-Protocol-for-DNA-Extraction-from-Staphylococcus-Aureus-4999.html
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