low plasmid yield - (Jul/31/2007 )
HI,
i am working with gram +ve bacteria, B.thuringiensis. i am trying isolate plasmid DNA for my genome walk experiments. with 400 ml culture i got only 36 micro gm of plasmid DNA. gel showed 1 neat band of 20 Kb. can any one suggest how to improve the yeild of plasmid DNA. i did not see pellet in the cenrifuge tubes but still went ahead to resuspend with TE buffer. and run sample on gel.
thanxs in adavance.
kemist
well i would suggest you to separate your extraction in 8experiments of 50ml each, without column, but standard alkaline lysis method, followed by PEG/NaCl purification overnight.
Moreover, after an overnight culture, add chloramphenicol to your culture (reduce RNA).
That will i suppose increase yield as i'm supposing bad cell lysis.
inaddition to fred_33's suggestion, I would add that inocculating your larger culture with cells from a fresh miniprep culture also helps. Growing your cells at a lower temperature also helps. I am unsure how large your plasmid is, but growing your cells (slowly) in a richer medium (like SOC, or adding 0.4% glycerol) can help.
try growing a small 10 ml culture through the day - 8-10 hrs and use it to inoculate a 400-500ml culutre overnight. May be this helps.
thanxs for all the suggestions, i am going to try all the methods n see.
i am growing the bacteria (Bt) in LB media witout antibiotic selection (plasmid doesnot have markers), usually overnight , 200rpm.
i have this particular strain, we are having tough time getting enough DNA for genome walk experiments.
and these bacterial strains have plasmids ranging from 2Kb to 200 Kb.
any more suggestions??
thanxs
kemist
This doesn't sound good. I have to ask, does the plasmid have selectable characteristic that will keep it around? Something that gives its host some kind of growth benefit?
A plasmid very much an extra burden to the cell (the bigger the plasmid the more of a burden it is). Only a selectable character of the plasmid keeps in the host population. Otherwise cells who by chance do not inherite the plasmid will grow faster and will eventually take over the culture. Resulting in poor if none existent plasmid yeilds.
I would strongly recommend that you grow your cells in SOC at 25-28 Celsius. A rich medium reduces the selective advantage a plasmid free cell over one which does. And the low temperature, reduces growth rate, again reducing any selective advantage a plasmid free cell might have.
This doesn't sound good. I have to ask, does the plasmid have selectable characteristic that will keep it around? Something that gives its host some kind of growth benefit?
A plasmid very much an extra burden to the cell (the bigger the plasmid the more of a burden it is). Only a selectable character of the plasmid keeps in the host population. Otherwise cells who by chance do not inherite the plasmid will grow faster and will eventually take over the culture. Resulting in poor if none existent plasmid yeilds.
I would strongly recommend that you grow your cells in SOC at 25-28 Celsius. A rich medium reduces the selective advantage a plasmid free cell over one which does. And the low temperature, reduces growth rate, again reducing any selective advantage a plasmid free cell might have.
HI,
the plasmid have insecticidal toxin genes n produces insecticidal proteins in this bacteria. i want to isolated enough plasmid DNA n see if i can fish out any toxin gene by genome walk.
we grow Bt at 30 C, is SOC media rich than LB?
yes, SOC is richer then LB. SOC has about 2.5x more typtone (free amino acids) then LB. (Thus more then double the price).
Moreover, after an overnight culture, add chloramphenicol to your culture (reduce RNA).
That will i suppose increase yield as i'm supposing bad cell lysis.
Fred_33
i am going to try the method you suggested, where can i get the protocol/KIT? plz let me know
kemist