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.
Hi again!
Thank you all so very much for the suggestions. There are so many ways to do this that specific names/protocols known to work well, like you all gave me, is truly appreciated! Elongating some parts of the protocols is a good idea; I am still rather new at this so I tend to follow the recipes to the letter; knowing I can play a bit with time/certain quantities is nice.
Sadly, I do not have the budget for enough of premade kits, but since some of them have their components and protocols listed once you get one, I should get one or two and see if I cannot "replicate" it.
@ Marcela: the lab next door has Chelex-100! I read a bit, and seems a good thing to try, thanks! :) I only do 16S, so it might be enough, and it's quick to try. If not, then I'll try the kits.
Thank you all again :)
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