Alkaline Lysis Mini prep smeared on gel - (Nov/15/2010 )
I am having a difficult time figuring out why my mini preps looked like bright smears on a gel. My classmate had the same problem. I have performed sucessful alkaline lysis minipreps in the past using the same technique. The reagents are about two months old. I did not use a kit. I am trying to extract pUC19 and pMALp2x from transformants from September. My classmate and I picked from the same plates. I spec my samples and averaged about 4000ng/uL. I appreciate any suggestions and tips!
Did you perform an RNAse digest?
Did you prepare the NaOH/SDS solution fresh, and how long did you incubate with this solution before adding the acetate solution?
what kind of e coli strain are you extracting DNA from?
Some strains (Especially expression strains) retain endonuclease function, which makes getting good plasmid DNA difficult.