what's wrong? Plasmid digestion produce only smear - (Aug/04/2005 )
When digest the plasmid which is minipreped by Qiagen kit, after running the gel, only the smear can be seen. What's wrong?
Yeah yeah I had the same problem too esp when I try to digest a recombinant plasmid that I obtained from clones. I can only get smears and nothing else. Anyone have the same prob? 
many buffers must contaminated by some people
Hmm dont think it's due to the contamination of buffers as it only happens when I digest plasmid that I purified from bacteria, esp if the plasmid is a recombinant plasmid as a product from ligation and insertion into bacteria. I use the same buffer for running all my gels, and the rest turn out to be ok. Any idea why?
Then it must be your preparation problem from bacteria. Either your buffer for that has problem or your operation problem. suppose your technique and knowledge is good enough. then: 1) check buffer, 2) consider to remove toxic material when you prepare the DNA
[quote=-YS-,Aug 5 2005, 07:43 PM]
[quote=shuiping,Aug 6 2005, 06:34 AM]many buffers must contaminated by some people
Hmm dont think it's due to the contamination of buffers as it only happens when I digest plasmid that I purified from bacteria, esp if the plasmid is a recombinant plasmid as a product from ligation and insertion into bacteria. I use the same buffer for running all my gels, and the rest turn out to be ok. Any idea why?
[/quote]
i am isolating recombinant plasmids by normal alkaline lysis technique. I am having no problem with the smears
try giving a RNase treatment before running the samples. I add RNase into the loading dye and incubate the samples for 15mins at 37C before running them. It should work.
Regards
Prajwal
Try using Recombinase minus strains. May be XL1Blue or Stbl2 should help.