My research entails extracting Bacterial DNA from patients' samples (plasma and csf) as I explore to use molecular techniques to identify bacteria indicated in non-traumatic coma. As most bacteria that cause this are gram positive which have thick cell wall, I see most papers adding modifications to the protocol so as to break down the thick cell wall, anyone done such before? Kindly assist
Dear Ian,
you can avoid this long prot-K+Lys digestion by increasing the sample temperature to 65C for one hour. I mean, perform the digestion instead of at 37C at 65C for one hour, and then continue with your protocol.
IT worked nice with gram positive bacteria from insects.
Dear Ian,
you can avoid this long prot-K+Lys digestion by increasing the sample temperature to 65C for one hour. I mean, perform the digestion instead of at 37C at 65C for one hour, and then continue with your protocol.
IT worked nice with gram positive bacteria from insects.
Hi
These long procedures only are necessary if you're working with yeast, parasite cysts or samples as tissue. For most purposes the use of proteinase k combined wiht a good lysis buffer will be enough to obtain cell lysis with Gram positive micro-organisms.
best regards
Hi Ian,
I've been extracting bacterial DNA from environmental samples using proteinaseK+lysis buffer at 65 degrees, but I found that optimal digestion is obtained through a combination of mechanical lysis (glass or zirconia/silica beads), proteinaseK at 65 degrees, and Ready-lyse Lysozyme (Epicentre). The combination of mechanical lysis + ready-lyse lyzosyme nearly tripled the amount of extracted DNA. Therefore, although I haven't investigated the effect that this had on the taxonomic diversity of standard vs improved extraction, the higher amounts of DNA suggest that some of it was inaccessible with simple pK+lysis buffer extraction.
Hope this helped.
Cheers
My experience of gram positive bacteria is with mycobacteria, which are hard to lyse. We needed to treat with lysozyme (final concentration ~10 mg/mL) 3 hr at 37 degrees C, then SDS (final concentration 2.8 - 3.6%), then proteinase K (final concentration 200-350 micrograms/mL overnight at 50 degrees C. To save time, I think that Christopher Sheridan's suggestion is definitely worth trying.
Hi Ian,
I've been extracting DNA from bacterial cultures (plant growth promoting bacteria) and other kind of bacteria using Wizard Genomic DNA Purification Kit (Promega) and I did not use Proteinase K and Lysozyme for gram + and -. The results indicated DNA concentration ranging from 50 to 700 (ng/uL).
Best regards
Mechanical lysis, without harsh chemicals or proteinase K can do the job in about 5 minutes if the mechanical lysis is performed using micromotor-driven disposable cartridges sold by Claremont BioSolutions. The OmniLyse(tm) device effectively lyses any cell type. The PureLyse(tm) device performs both lysis and extraction of DNA. Both perform their sample prep in less than five minutes. See www.claremontbio.com.
My experience of gram positive bacteria is with lactic acid bacteria. We needed to treat with lysozyme (final concentration ~10 mg/mL) 3 hr at 37C, then SDS ( 4 %), then proteinase K ( 200 micrograms/mL 30 min at 50 C.
Or add lysozyme (in TE) for 15 min at room temp followed by 4% SDS as described by Mohammed) + Proteinase K @ 55C for 1 hour..Then continue
Hello Christopher and Anne, could you please tell me if the enzymatic steps can be included into a commercial DNA extraction kit (UltraClean Microbial DNA Isolation Kit, MOBIO Labs) which uses a combination of heat, detergent and mechanical forces (microbeads). Can you suggest a reported protocol?
I find freeze/thaw cycles (3) plus use of a sonic dismembrator works for me and avoids glass beads, at least with yeast and bacteria.
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