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
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