smear in miniprep DNA - (Dec/15/2006 )
I am having problem in my miniprep DNA preparation which I never had before.
1. I am getting very low yeild and on digestion all I can see in the gel is a smear.
2. Even If the DNA yeild is O.K than on digestion I see only a smear.
I tried changing the competent cells, R.E buffers, different enzymes, but no solution. My previously prepared plasmids (before I had this problem) give good bands under the same reaction conditions. I guess something wrong with my miniprep DNA. I am using Promega Kit and following their protocols.
just to confirm....
1- Have changed the strain of cell you are using? Are you using are proper cloning type strain as oppose to an expression type strain.
2- Is your solution 2 (Alkaline lysis solution) old or has been left open and uncapped for long periods of time? Alternatively, is your solution 3 (neutralising solution) at the right concentration?
This sounds like DNA degredation (perhaps by bacterial endonucleases... though this would only happen in expression strains)but the low yield may point towards problems in alkaline lysis solution.
I agree with perneseblue, Sounds like DNA degradation.
Check the bacterial strain may b it has some nuclease eluting with the DNA. And also check if u added RNAse in ur resuspension buffer.
I agree with perneseblue and Scolix
Thanks for the replies.
But as I told you I am using Promega Kit and following their protocols so I do not think its the problem of the solutions.
I have checked my strains. I am using JM109 and in th same cells till now I had no problem with my miniprep DNA and I used the same Kit.
Anyway I am redoing it and will change my lysis solution.