plasmid digestion gel - (Jul/29/2008 )
I did a ligation and a transformation and got a few, tiny colonies in my plates of ligated DNA. Positive control was good and the negative control did not show any colonies. I further cultured these colonies in LB broth with Amp and extracted DNA. I digested DNA for 3 hrs and ran on a gel. I only obtained a smear and no clear bands. I digested my PCR product of the insert also at the same time and ran on the same gel. I saw a similar smear for that also.
Now I am trying to understand what went wrong!, whether the colonies I observed on the plates were not what I expected? or whether I used too much restriction enzymes for the digestion? or some other problem? I digested (3 hrs) my vector using the same enzymes a few days before and I did not have any problems.
If any of you have an idea about this observation please let me know
Thanks
well, there are two possibilities
1 - a very bad miniprep preparation, followed by a bad PCR (typically from using too much DNA template)
2 - the ligation has failed and you are not seeing anything because there is nothing to see.
May I ask, how confident are you that the miniprep DNA is okay? How many different preps were made and resulted in a smear?
If possible can we see the picture of the gel, it would help abit. Could you run a undigested plasmid DNA to confirm that there is plasmid DNA present. And just to confirm, the PCR being run is across the junction between insert and vector
I made 8 preps I tried to digest 4 all were smears. I have attached my gel images here. I appreciate your suggestions. Thanks
Other possibilities are that your enzyme is exhibiting star activity, that one of your ingredients is contaminated with a DNAse or with another frequently-cutting endonuclease.
What restriction enzyme did you use, and how did you use it?
If your protocol is certain that is you frequently use minipreps and these restriction enzymes I think the logic answer is that ligation has failed! Don't lose time and do another ligation. I think you have the optimum 1:3 vector: insert ratio....