smear when cutting plasmids previously checked - (Nov/14/2007 )
When I try to cut two different plasmids (different vectors with the same insert) after miniplep or midiprep I always have smear and never the band or bands (in case of double digestion). Both plasmids had been checked with sequencing before making the glycerol stocks. When I run the plasmid uncut it seems OK. I have checked different enzymes from different companies and none worked. I sterilized the water again and no result. The two glycerol stocks come from bacterial strains from different origin. One or two years ago I did not have problem cutting the same plasmids from the same glycerol stocks. I considered plasmid production a very simple thing and now I have stuck.
I would appreciate any ideas!
Thanks in advance
let me get this straight
-raw DNA looks fine on the gel
-digested DNA looks smeary
-have tried different enzymes by digest still looks smeary.
Have you tried using a new vial of restriction digest buffer? Perhaps the buffer has been contaminated. A silly question, but is the gel isn't overloaded right?
Do you percipitate your restriction digest prior to running it on the gel? Could this be a case of running the gel too hard (if the restriction digest was direct loaded and run on the gel)
Is it possible the buffer the DNA is stored (maxi or midi) was some how contaminated and addition of the buffers for restriction digest, activates others enzymes degrading the DNA. COuld you precipitate the DNA and redissolve in some other solution may be TE.
-raw DNA looks fine on the gel
-digested DNA looks smeary
-have tried different enzymes by digest still looks smeary.
Have you tried using a new vial of restriction digest buffer? Perhaps the buffer has been contaminated. A silly question, but is the gel isn't overloaded right?
Do you percipitate your restriction digest prior to running it on the gel? Could this be a case of running the gel too hard (if the restriction digest was direct loaded and run on the gel)
I used different enzymes with different digest buffers, so I don't think that is something wrong with it. Now, concerning the precipitation, I just do a small spin prior running it on the gel. But I've done it many times before and it was OK.
Thanks for your interest
Check how you're doing your DNA preps. I've had this happen to me many times, and almost every time it's because I didn't use clean enough DNA. Some bacteria contain extra Dnases that will cut up your DNA during the digest resulting in a smear or a complete dissappearance of your DNA. check what type of bacteria your vectors are in and use either a Quiagen miniprep (with all of the wash buffers provided) or a homemade prep that includes a phenol extraction.