If I were to use a certain volume of mycobacterial culture (atypical mycobacteria) directly from a liquid broth and subject it to conventional DNA extraction kit protocols using silica membrane spin columns, would the mycobacterial cell wall interfere with the bacterial cell lysis step during the extraction process? I have read that their cell wall is a strong protective barrier, so the cells have to be ruptured first by boiling or using glass beads. I could be wrong of course.
If you are working with only cultures, normal thermo-chemical lysis would be sufficient. For confirmation you can perform one extraction with kit protocol and another one with additional pre-boiling. Perform qPCR and if Ct values match, it means the recovery are comparable between these two techniques.
Wall cell lysis is necessary in any form, chemical or physical, you have to check in the kit if the method is adequate for fungal walls, walls could be a important barrier to obtain good results in DNA quality
Hi, I think the best approach would be dependent on the use you have for the DNA. I like mechanical methods for difficult to open organisms (bacerial and fungal spores, would reccomned MoBio Powersoil for a good general purpose method) but the down side is that the DNA is sheared and will only be a few kb in average length.
Spin column kit already contains lysis buffer (SDS and proteinase K) that should be added before boiling for complete cell wall lysis. I used similar kit for capsulated fungi ( cryptococcus yeast) and it is perfect actually
so, yes boiling is very important especially for capsulated organisms
Hello there,
You know, I presume that, the distinguishing characteristic of all Mycobacterial species is that the cell wall is thicker than in many other bacteria, which is hydrophobic, waxy, and rich in mycolic acids and mycolates. The cell wall consists of the hydrophobic mycolate layer and a thick peptidoglycan layer held together by a polysaccharide, arabinogalactan. Therefore, boiling is going to just get rid of your waxy materials without affecting your thick cell wall. Using glass bead is a bit harsh since it shears your DNA and might render it useless for your downstream manipulation. I suggest using sonication on ice for different amounts of time and than find out at what setting and duration of sonication you can get the best DNA isolation from your kit.
Chris
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