Help in RE digestion of vector - (Dec/10/2011 )
I did RE digestion (Double digestion) of my vector Using fresh newly bought Enzymes
two times I tried both times I was getting a Smeared band after 3 hr of digestion at 37 deg
The band is having high intensity at the top and fades away slowly
the problem is that i am not able to decide where to excise the gel and use as linear vector
as the faint as the band size certainly different (bigger) from that of expected ones
I am adding RNase to my plasmid vector during extraction by alkaline lysis method
and the gel image of the isolated plasmid vector was very fine with only 3 bands (circular)
Give me some ideas on this aspect which i can try so as to avoid the problem.
Please give us further information.
How did you set up the reaction?
Which enzymes are you talking about?
The band fading is probably due to migration of dye toward the negative electrode. Add dye to the running buffer at the positive electrode to avoid this. A picture of your gel would help.
If you see fade smear kind pattern then look like your DNA is degrading. Check water you used to digest or reduce the time of digestion.
And it will be nice if you tell us more which enzyme are you used and how much DNA used for digestion. if possible upload the gel picture
3 hour is a bit too long. Sometimes long hour digestion did give me smearing result. In my own experience, to digest plasmid, 45 min- 1hour 30mins is long enough. You could try to use a shorter time (1 hour) to see what will happen. Actually varying digestion time is a part of optimization for RE digestion as well.
Often smearing is due to overload of DNA on a gel. You may be trying to digest too much DNA, or to load too much DNA on the gel.