Dear members!
I have two questions/issues:
1) Does anybody know how to solve the problem with FD Enzymes for preparation of cloning vectors?
I make restriction before cloning with FD XbaI + FD HindIII + FD Green buffer according to protocol. I tried it few times of digestions: 2h, 4h, overnight
After cloning I get my target inserts but in very low effective. Most colonies contain uncut plasmid.
2) Also I use FD DpnI enzyme to reduce bacterial backbones after PCR when I need to clone my amplicons in another plasmid. So, I face with the same problem in this case too. Electrophoresis doesn't help to separate it properly after digestion
As per the size of your construct. Try to increase the gel percentage for good separation and then gel extract both the insert and the vector. And for efficient digestion use a slight more amount of RE, since with the progression of time, enzyme losses its activity slowly
Both enzymes are cutting so part of the solution may simply be the size of the insert. It is smaller than the plasmid so will trap less ETBR and be a less intense band just because it is smaller. It may be that some of the cut sites were damaged in ligation or that the plasmid is a mixture of inserted and not inserted in spite of the selection method
I usually purify the bands of vector and inset from the agarose gel. You can also add alkaline phosphatase when digesting the vector, so as to decrease the chances of vector self circularization.
I concur that I think you are getting good digestion. As Paul Rutland mentions, you expect the band to be less intense. Also if you look carefully at the plasmid band, it is not showing two different sizes. However even if you are getting 99% digestion of the plasmid, the remaining 1% will give you lots of colonies upon transformation.
If there is another restriction site in the vector that lies between the HindIII and XbaI sites, you could digest with that enzyme after ligation (assuming it is not present in the insert). That way any ligated plasmids with the insert will have lost the site but any background plasmids will get cut by that restriction enzyme. It is a good way to reduce the plasmid background if the restriction maps work in your favor.
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