I want to get rid of a restriction site for an enzyme in my vector. At first I tried to mutate the site by site directed mutagenesis but somehow it did not work. Then I adapted a straight forward approach to digest the vector with the RE in the question and then chew out the sticky overhangs by Mung bean nuclease digestion. I gel purified it and and religate the blunt ended vector. Unfortunately after screening few clones I see that they have same sequence as original vector. However the Gel showed that the digested vector migrated differently (slightly slower) when compared to the undigested control suggesting RE digestion worked. I suppose there might be two problems:
1. Mung bean nuclease digestion did not work efficiently and hence there were some sticky ends vector which have higher ligation efficiency when compare to blunt ended vector.
2. The RE digestion was not complete so some undigested vector were there which some how did not get seperated properly on electrophoresis.
Therefore I decided to dephosphorylate the RE digested vector before mung bean digestion so that even if the MUng bean digestion is incomplete, the dephosphorylated sticky ends will not religate. But I have a doubt that whether the dephosphorylated overhangs are still substrate for Mung bean or it only binds to phosphorylated overhangs. In my opinion this should not be phosphorylation dependent but please provide your suggestions.
Thanks
Prafull
Hi there,
As Mung Bean Nuclease acts efficiently on both 5' and 3'ends (3'OH), I would anticipate it works fine on dephosphorylated 5' too.
Hi :)
Where is the restriction site located related to your insert? Do you need the surrounded area? If at MCS, maybe it is easier to cleave the site out with other flanked restriction sites up or downstream combined with a dummy insert. So you get bigger fragments and be able to separate them more easily.
I would also check if there are other restriction sites that result in having the same sticky ends.
If you can leak some more details on the positioning of the restriction site its easier to think about it.
If all you want is to get rid of a restriction site that leaves 5' overhangs, you can also "bluntify" your vector by filling the sticky ends with Klenow polymerase. The problem with mung bean nuclease is that it will cut your plasmid at any site that is nicked, which is likely to occur if your plasmid prep is old.
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