SDS vs. CTAB vs. Sarkosyl - cell lysis/DNA extraction (Nov/24/2008 )
Hi there,
I am about to go crazy. I am at a point where I read too many protocols and papers about extraction buffers that my head is spinning and I am not sure anymore which buffer to use. HELP!
My goal is to extract DNA from cyanobacteria (and soil later on). The most critical point right now is cell lysis (especially for cyanobacteria), so I was trying to figure what lysis/extraction buffer to use. But I am a bit confused because I found several combinations: SDS or CTAB or Sarkosyl or SDS+CTAB or Sarkosyl+SDS or Sarkosyl+CTAB.........do you have any experience with those extraction buffers? Is one better than the other? Which combination works best for you?
Some buffer also had PVP or DTT (or 2-mercaptoethanol).......how do those reagents contribute to breaking cells or extracting DNA? [sorry, I feel I should know this, but I don't......all I know is that DTT is a reducing agent for disulfide bonds in proteins, but I don't how it helps in the extraction.]
I would be grateful for any advice and help.
Cheers!
Do you have a little bit information about Cyanobacteria? How is it different from E. coli extraction (e.g. what is special about the cell wall etc.). For example I work with mycobacteria and the cell wall is much tougher so we have to use different methods because buffers that break open E. coli do nothing to mycobacteria.
Did you look if kits for DNA isolation are available?
I don't have any experience with cyanobacteria, but CTAB and SDS is usually sufficient for plant cell walls (yes, I know cyanobacteria aren't plants). Soil complicates things a bit, lots of soluble materials and extraneous protein from bacteria etc. Sarkosyl and CTAB are just detergents with increased strength over the usual SDS, triton, etc.
PVP is a long polymer that binds to polyphenolics, one of the major components of cell walls, effectively removing them from solutions. DTT and/or 2-ME are denaturing and reducing agents that will denature DNases and RNases along with other proteins, stopping them from digesting your ribonucleotide of choice before it is purified.
Did you look if kits for DNA isolation are available?
cyanobacteria can form biofilms and surround themselves with gelatinous sheet (polysaccharides) that can interfere with the enzymatic digestion. Breaking them apart is an important factor to make lysis possible (I am currently trying freeze-thaw cycles....and I was planning on trying beads as well). But chemically I am not sure what to use (to continue lysis).....if either CTAB or Sarkosyl or SDS or a combination of all three.
There are kits for soil samples, but I haven't seen one for cyanobacteria.....I like the idea of having a own protocol because it gives you the chances to influence the reaction and to modify them if necessary (well, and it's a bit cheaper).
Please let me know if I didn't answer your question.....Cheers!
PVP is a long polymer that binds to polyphenolics, one of the major components of cell walls, effectively removing them from solutions. DTT and/or 2-ME are denaturing and reducing agents that will denature DNases and RNases along with other proteins, stopping them from digesting your ribonucleotide of choice before it is purified.
Thanks for your input!
Do you think it matters if one would use a combination of SDS+CTAB or SDS+Sarkosyl or doesn't it matter? It seems like that some people prefer one or the other.
Cheers!
We use bead beating for mycobacteria since this is a good way to break up the tough cell wall of those bacteria. If you have beads and a bead beater, I will be happy to send you a protocol (just need your email).
However, the problem with beads is that they will also destroy the genomic DNA. Not completely but you won't have complete genomic molecules anymore. If you want to do southern blots for example, you want to have full length genomic DNA.
I can also send you a protocol to for isolating genomic DNA without beads. That will give you the full length chromosomal DNA. During the end of the protocol the DNA falls in strands out of solutions and you can easily fish it with a pipette tip. None of the buffers you mentioned above is used in that protocol. So you don't have to worry about it
Let me know if you want to try any of those protocols.
i haven't worked with them in over 30 years (they were still called blue-green algae then) and we were isolating proteins, but we used to use an "eaton press" to break open our cyanobacteria (unicellular: anacystis nidulans, and filamentous: anabaena flos-aquae).
the eaton press (named after the designer, a professor at brooklyn college) is a modification of the french press (which you may also try), it pushes frozen slurry through the hole rather than liquid.
i have only seen them used in brooklyn college and the university of missouri at columbia (i brought one there). i don't know where they may be sold but you may be able to contact someone at brooklyn college to determine availability, if you are interested.