Genomic DNA Isolation kit for Gram Positive? - can you recommend one for L.lactis? (Sep/20/2006 )
Hello Gurus,
Do you have experience in isolating Genomic DNA from Gram positive Bacteria ?
I have found many kits that would work well on other organisms.... but none is really efficient with Lactococcus lactis (IL1403)
Any help ?
promega's wizard prep, gram positive version of the protocol, we use lysostaphin to break up the gram + cell wall, for S aureus...there are a few minor adaptations to the protocol
there is also a homebrew method involving lysostaphin and proteinase K, with a P:C step to purify
I can send you either protocol if you like? your lactococcus may prefer lysozyme to lysostaphin, but we find in our bugs that lysozyme is not an efficient lytic agent at reasonable concentrations/incubation times
there is also a homebrew method involving lysostaphin and proteinase K, with a P:C step to purify
I can send you either protocol if you like? your lactococcus may prefer lysozyme to lysostaphin, but we find in our bugs that lysozyme is not an efficient lytic agent at reasonable concentrations/incubation times
I would be very thankful if you kindly send me your protocol.
I have been using Lysosyme at very high concentrations (1mg/ml) to lyse my lacto, and that is making Bradford det. tough.
please do send
thank you for your recommendations
I really appreciate that
I would contact promega immediately.
copper, before you get excited about buying a kit, you may want to purchase a small amount of lysostaphin and see if it lyses your cells better?
as far as changes to promega's protocol, I was unable to get decent lysis following the instructions as printed. this is what I do, that works pretty well on S aureus and you may want to try it? I will write it as adaptations to steps in page 16 of this pdf
(it's the bacterial version of the protocol, if for some reason you can't follow the link)
CHANGES:
step 2: resuspend in 150ul EDTA, 50mM
step 4: add 15-30ul lysostaphin, 1 mg/ml (NOT 10 mg/ml); incubate 15'-60' at 37. do NOT centrifuge after this step, you won't separate anything out. you will know if lysis has been efficient because the contents of the tube will be clear
step 5: add 500ul nuclei lysis solution; do NOT pipet here, just invert 5-6X
everything else is the same. hopefully you may be able to adapt this to your needs? also, I use my DNA for PCR and I find that things are a bit more consistent if the final DNA pellet is resuspended in 10mM tris.cl, pH ~8 to 8.5, instead of the TE supplied in the kit (so you dont' have to worry about the EDTA later).
good luck!
I really appreciate your help
thank you very much
I will try that and let you know if that works for me.
thank you again
hi, i used DNeasy® Tissue Kit (Qiagen, Germany) for my Lactobacillus spp. and it works well for my sample.
Just that u will need to prepare a enzymatic lysis solution with lysozyme.
Connice