I am following alkaline lysis protocol very carefully, with special attention to the correct pH of all the three solutions - TE with glucose, NaOH with sds and K-acetate with glacial acetic acid pH 5.2, etc. On my gels after restriction linearization (carefully chosen no to cut inside the gene) I get only small fragments that are somewhat smeared. I also did a phenol:chloroform extraction to clean up the DNA before digestion and remove any residual protein. Are there some tips to getting this right? I let the NaOH/sds with cell pellet rest on ice for 2 minutes before using the potassium acetate solution, and I did not vortex at all. Is that 2 minutes too long? Some protocols do not recommend this 2-5 minute incubation.
thank you! - Amy J.
These videos show the protocol I am using
https://rahulpatharkar.000webhostapp.com/tag/plasmid
It may be I am using cells that went into stationary phase too long and suffered plasmid degradation.
Amy, the first thing is to confirm that you actually have good plasmid DNA after your miniprep. If you run undigested plasmid on a gel, what does it look like? Do you have good strong plasmid bands? If you have plenty of DNA when uncut, but it seems to be smeared or missing after restriction digest, then you likely have some issue with contaminating nuclease. On the other hand, if you don't have lots of plasmid DNA in the undigested sample, then the issue is likely with your miniprep protocol (assuming your strain is good and actually carrying a plasmid).
But to answer your direct question, when you add the NaOH-SDS the solution should go from turbid to quite clear. If that has happened, then it is ready to go. There is no need to wait but it shouldn't hurt.
thank you! my main question has been how long to let it sit between highly basic and then the pH 5.2 k-acetate
The answer is that once the solution turns clear it is done, but a few minutes on ice won't hurt at all. I frequently did 48 mini preps at a time and it certainly took 5-10 minutes or so by the time I finished adding solution 2 to all tubes before I got around to adding solution 3. That step is not likely to be the source of your problem with smearing.
You are welcome, let me know if you still have smearing problems as there may well be issues other than the miniprep procedure.
And what are we both doing on here on Dec 25?? :)
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