Separate cutting plasmid from uncut? - (Aug/10/2007 )
I want to remove promoter from pBI121 . pBI121 has 14758 bp and promoter 835bp.Of course, after digestion
all of my plasmids don't cut and some of plasmid remain uncut.So... Can I separate cut and uncut plasmid on gel? what concentration of gel?
and if I don't do that, this mixture could become problematic for me?
The last question is : How I can increase my cutting plasmids ratio?
Thanks in advance
The separation between 800 bp and 14Kb is possible using 0.5 % gel. The separation of 14.7 Kb and 14 Kb is nearly impossible giving the difference is so small. Your best bet will be to cut the plasmid as completely as possible by prolong the reaction time (overnight) or repeat the restriction procedure once, then ligation, transform bacteria with electroporation using good competent cells (becasue large size), then screen for positive clones. You can use unligated DNA as a control to estimate how much uncut is present in the mixture.
In addition to genehunter's suggestion, run the gel for a much longer period and slowly so that you could separate the completely digested vector from partially digested.
What do you mean increase my cutting plasmid ratio?
what you can do is to cut by enzymes which are active on borders and inside the promoter. That will drive more oponed plasmids which reduces the self religation.
Thanks all for your helpful suggestion.
Increase the cutting plasmid ratio means I have more cutting plasmid than uncut ones.