Recently I read in brief about a method of bacterial DNA extraction where the researchers were using lysis to break open the cells and then somehow managed to concentrate PCR inhibitors in the same tube itself, before using the lysis solution as a template for their PCR. They called it the PURE method DNA extraction. I have not been able to find any protocol which manages to do something like this.
Have you heard about such a protocol used by yourself? Could you please forward me some details?
which type of bacteria? for example for Gram Negative (E.coli, Salmonella,..) you can just boil the broth for 5 min then shortly centrifuge it and then use the supernatant for the PCR. we did it once that we took the colonies from the plate directly into the cycler and it worked. But for Gram positive it is more difficult. For TB it extremely difficult but boiling protocols are also used.
Many years ago people used to use Chelex-100 beads to remove impurities /inhibitors from lysed bacteria / cell extracts. With gram positive cells lysing is much more difficult as Amr has said. People have tried using a combination of microwave,enymatic digestion and Chelex-100 for gram positive bacteria. See links below or Google Chelex and bacterial DNA extraction.
http://ac.els-cdn.com/092325089290107Y/1-s2.0-092325089290107Y-main.pdf?_tid=c6a33ae2-da77-11e3-bca2-00000aacb362&acdnat=1399969552_6eef7625d876ede8b8aa8c681001a105
http://download.springer.com/static/pdf/178/art%253A10.1007%252Fs10482-010-9462-0.pdf?auth66=1400141810_f82198b6e91f5f7f37186d15e4492778&ext=.pdf
Ian
Dear Amr and Ian,
Thank you for your replies. I am aware of the lysis method of DNA extraction. But this is a very crude method of DNA extraction. I have also come across some commercially available kits that give DNA containing supernatant with 15-20 mins of heating.
The protocol I am looking for is likely to be similar to these, just that along with the heating them, there is some ingredient that is capable of holding onto common inhibitors. I do not mind commercially available kits as well as along as they can save me time.
Thanks,
Ameya
I appreciate this is an old thread, but Erik Zinser adapted the use of heat lysis for qPCR of environmental marine cyanobacteria with great success in qPCR assays to quantify these bacteria from field samples. In brief, 100 ml of seawater is filtered onto a 0.2 µm filter then the filter is essentially heated in 10 mM Tris for 15 min at 95 C, then a few µl is added to the qPCR reaction. The heat lysis should inactivate most or all enzymes like DNAses. I'm not sure what types of samples you are working with, so certainly there could still be inhibitors that are inactivated by heat by this method.
Article Prochlorococcus Ecotype Abundances in the North Atlantic Oce...
I also recently came across this nice paper that systematically tested heat lysis on E. coli.
Article Performance Evaluation of Fast Microfluidic Thermal Lysis of...
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