Problem with Plasmid Extraction

[Trial&Error]

Hello everyone, i am happy to join this nice forum.

I ve done an alkaline SDS extraction of plasmid from hospital acquired Proteus strains an environmental E. coli strains

I prepared the reagents manually, and faced a problem with pH adjustment of the lysis buffer (2 pellets NaOH + 5 mMTris + 1 mM EDTA + 4 g [!] SDS in 100 ml water).
According to the protocol, pH should be 12.4. When I first prepared the solution, pH was around 10.5, I added NaOH (dry) , but the maximum pH I could reach was 11.6, adding any more NaOH decreased the pH !!

When electrophoresis was done, only the marker is seen and NO plamids from about 30 strain were detected.

Any help to overcome this problem may be life-saving

[admin]

Hey Trial,
which protocol are you using for DNA extraction it seems a bit off? Have you tried any kit based methods?

[Trial&Error]

This is too late reply.

But the point is I left this experiment to some friends, just yesterday my friend told me that they repeated the electrophoresis run, just electrophoresis not the extraction, and he surprised me that there were bands in most strains.

The difference was (1) in the concentration on agarose, I used 0.8% and they used 1.6%, and (2) I stained the gel in less ethidium promide (we put ethidium promide with the agarose during preparation).

[admin]

Happy it worked!
Yeah thanks Trial, that is a good idea they did your DNA probably ran of the gel if you ran it too long or they were small DNA sizes.

thanks for letting us know what happened and welcome the forums my friend