Hi:
I was wondering if running a phenol:chloroform extraction before an alkaline lysis separation of plasmid dna from genomic would be of benefit (ie cleaner sample free of protein etc) or would this hinder the standard alkaline lysis protocol?
thank you! - Amy J.
Hi Amy,
It is the standard Alkaline lysis followed by potassium acetate neutralization method, which allows plasmid DNA to reanneal quickly and stay solubalized, while chromosomal DNA, lipid and proteins form complex with SDS and potassium acetate, which is removed by centrifugation. Be very very careful about the Pottasium acetate solution, particularly the pH ( 3.0 M, pH 5.0). Also, this solution is to kept at RT and used as such. Adding cold solution (kept at 4C) to the lysis mix may not give expected results.
If all the reagents and solutions are prepared exactly as recommended, you are expected to get nice plasmid preparations, which are clean enough to be used as template in PCR after at least 10 fold dilution or directly for RE digestions. However, if you still feel that some amounts of proteins are left with your plasmid preparations (by a quick nanodrop assessment), you can purify them through Phenol:Chloroform extraction followed by Isopropanol precipitation or using a standard column based DNA purification kit.
Therefore, I think that doing a Phenol: choloroform extraction before Alkaline lysis is not going to make in particular difference. Rather, traces of carried forward organic solvents may in fact interfere with downstream alkaline lysis method.
best wishes,
SD
thank you so much! The protocol I am using calls for the potassium acetate to be at 5M, though and 5.2 pH. I am using the Maniatis protocol, from the Blue Book. Do you get better results at a lower concentration of k-acetate?
regards, - Amy J.
Hi Amy,
To make Alkaline Lysis Solution III, you need to add 60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid and 28.5 mL of nuclease free water.
The final solution is now 3 M with respect to potassium and 5 M with respect to acetate. Adjust the pH to 5.0-5.2 and you are ready to use it.
Best of luck.
SD
yes that is the recipe I used, and I was able to precipitate out about 100 ng of probable plasmid dna, and I checked the concentration with a Qubit flourometer. I used this DNA to run two pcr reactions with my gibson primers, and I got 30 ng/ul reading targeting 1 fragment of the plasmid and 201 ng/ul ! on the other targeted section of the plasmid. Used phusion taq and protocol, GC buffer. Running a gel now to confirm the results. thank you so much! The precipitate was a tiny visible pellet, but it was measured at 2ng/ul concentration in 50 ul TE after removal with a pipette tip and resuspension in the TE.
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