Hi!
I have been trying to extract DNA from anaerobic bacteria (both gram positive and negative) using the lyzozyme/proteinase K/sds/chloroform/isopropanol protocol, but have repeatly failed to get DNA.
I am using "wide" 16S primers for the pcr (a combination of 27F YM + 27F BIf + 27F CHl + 27F BOR and 907R), and test on a 1% gel. These primers have served me very well with aerobic bacteria (with a regular heat prep only). But with anaerobic bacteria, I seem to have hit a wall.
Any protocol suggestions that are known to work with those hard to crack open anaerobic bacteria, especially when cells are only available in smaller quantities? Bullet blenders?
Thank you for your help
Lodo
first of all check your bacterial culture shold be viable and fresh. Second check the temperature of your solutions. Third try longer time with some steps as the cell wall may need more time or larger amount of enzyme for destruction
Dear Gunes-Helene. I work with C. difficile. For target amplification and/or sequencing I use Chelex 100 resin with no problem, it is cheap and quick. For WGS I use commercial kit : https://www.fishersci.com/shop/products/invitrogen-chargeswitch-gdna-mini-bacteria-kit/cs11301
They have protocol for G- as well as G+.
Good luck!
Marcela
I've had good luck with the Qiagen DNeasy blood & tissue kit and using the protocol for gram-negative bacteria. You can increase the incubation times from 10 to 30 minutes and that does increase yield.
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