Hi all,
I am struggling with exctraction of plasmids from some isolates of Klebsiella pneumoniae. I am using a manual extraction protocol that I am attaching in this message. Last week this protocol worked well and I was able to extract plasmids from my bacteria, but this week I tried the same extraction with the same steps three times on the same isolates and it did not work. I can't understand what goes wtong. I am unable to detect any pellett at the step 20 (after centrifugation with ethanol). It seems like I don't have plasmids or I lost them during the extraction procedure. But the protocol worked last week. I prepared lysis buffer solution and RNAase solution fresh everytime. The strange thing I noticed is that the phenol solution changed color from yellow to red-pink. So I don't know if this affects the results. The other strange thing I noticed is that the Mac Conkey plates I am using (from which I pick the colonies up for the overnight culture in broth the day before the extraction) changed color from red to some sort of "yellow". They have been streaked 2 weeks ago and I was wondering if maybe something wrong happened to bacteria..I am aware that they can loose their plasmids but after subculturing and subculturing them. Is it right?My plates were made from the stock of my bacteria. Is it likely they loose plasmids some time also in the same plates streaked from the stock?
Any suggestion would be really appreciated.
Thanks
I do not know what exactly is a reason by you can enhance the plasmid expression if you use a sub-optimal concentration of an antibiotic for which it is resistant. the use of such antibiotics will make sure it does not loses the plasmid and express it even more. just try it once.
Dear Silvia,
One problem i can find that is change of phenol solution color from yellow to pink. It means phenol got oxidized and so it can not be used anymore for palsmid extraction. Prepare the phenol solution freshly (hope you have protocol for that). Pour 1ml layer of Tris HCl (pH8.0) on top of the phenol solution to avoid oxidation and store it in an amber bottle at 4 degree celsius to avoid contact with light. If I am correct you should get the plasmid in your next experiment. All the best.
dear caprari,
1. as of provided information, its better to avoid the use of long stored subcultures for plasmid isolation (overnight culturing) as there is a chance to loss or mutation of the plasmid. from the stock: culture the bacteria on selective antibiotic containing media, enumerate and then use those cultures. (but don't store for longer time)
2. As prem suggested; always use fresh tris saturated phenol of ph 8-8.5. if you want to extend self life of solution & stabilization you can add 0.1% hydroxy quionine during preparation. this also acts an inhibitor for deoxyribonucleases.
All the best
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