I want to extract DNA from gram-positive bacteria grown in pure cultures. The pre-treatment recommended in the Qiagen DNeasy® Blood & Tissue kit handbook involves the use of 180 ul of an enzymatic lysis buffer, followed by 25 ul of proteinase.
Which enzymatic lysis buffer would you recommend? Does it have to necessarily include lysozyme?
I am not sure the Qiagen DNeasy® Blood & Tissue kit is appropriate for DNA extraction from bacteria grown in pure cultures. As the name of kit says: "blood & tissue". However, Qiagen has another kit called Pathogen kit.
Hi Miguel, Thanks for your answer. The kit works fine with bacteria, I´ve worked with it before on gram-negative bacteria. The protocole is actually described in the handbook, I was just wondering about the adequate pre-treatment...
Hi Frédérique, I use the same protocol than you. I prepare the enzymatic lysis buffer using:
20 mM Tris·Cl, pH 8.0
2 mM sodium EDTA
1.2% Triton® X-100.
20 mg/ml of lysozyme.
I mix Tris·Cl, sodium EDTA and Triton® X-100 without lysozyme and is stored at 4 ºC. For each assay, the volume required according to the number of samples is taken (+ 2,) and immediately before to use, lysozyme is added.
Handbook, page 14 and 45:
http://diagnostics1.com/MANUAL/General_Qiagen.pdf
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