Why freshly prepared alkaline solution II for plasmid extraction? - (Oct/05/2005 )
Hi,everyone.Why do we need to use freshly prepared Alkaline Lysis Solution II for plasmid extraction?
After performing plasmid extraction,I noticed that the remaining Alkaline Lysis Solution II is normally wasted.I usually prepare 25 ml of Alkaline Lysis Solution II,however,only 200 microliters of the solution is added into each sample. Therefore, each time I do plasmid extraction, the remaining Solution II is normally wasted. Is there a better way to prepare SOlution II?
What is the composition of that solution?
Also, you could always scale things down to say 1 or 2 mL.
i don't know that it's necessary to do it EVERY time...I generally make up fresh PII about every other week and it's all OK
a way to make it easier, make up each component at 2X concentration and mix just enough to use
oh, hey, I am assuming you are making the SDS / NaOH lysis solution?
Actually, although I agree that all the protocol books (Maniatis, etc.) do say that this must be prepared fresh, I don't think it's required.
I mean, most (all?) plasmid mini- and midi-prep kits from Qiagen, Promega, Invitrogen, et al. rely on a straight forward Birnboim & Dolly alkaline lysis procedure; the only modification is column-based recovery rather than precipitation. These kits sit in a warehouse or two before they arrive at the lab, then sit on your lab shelf while you use them up, and there's no problem with the NaOH/SDS solution...
Thanks for the help.
By the way,as suggested by aimikins,if i would like to prepare Alkaline Lysis Solution II one week before i start to do plasmid extraction,what would be the best way to store this solution?In room temperature or in cold?
I think you'd get a precipitate if it's stored in the cold, but if you just warm it up in a waterbath, it'll redissolve.
Still, if I had to guess, I'd just hold it at room temperature.
precipitate is caused by SDS in your solution. I store my solutions for preps on the bench for a month and i get good recovery.
but i'm questionning myself about your solution wasted... i think that SDS would inhibit any contamination by oganisms... you can autoclave your solution II for more security.
I'm not sure you need to worry about bacterial growth in a solution that's 0.2 N NaOH and 0.1%SDS, do you?
I guess the alkaline lysis soln neednt be freshly prepared. What should be kept in mind is that the vessel should be air tight cause the only problem is SDS getting precitated which ofcorse dissolves on boiling. Infact we use Qiagen's kit in which the soln II is provided and there is no specifcations of preparing it.
Hi make about 50ml stock of 10N Naoh and 10% SDS. Everytime you can dilute them ,NaOH 1/50th time and SDS 1/10th time to get 0.2N NaOH and 1%SDS.