In constructing shRNA plasmids, I want to shorten the size of the plasmids, so I want to use two restriction enzymes for restriction digestion, and then use T4 DNA ligase for complement and smooth connection. Does this cause the loss of several bases at the restriction enzyme digestion site? How can I avoid this?
I presume these two restriction sites are either the same or if they differ but generate the same sticky ends, or if they both leave blunt ends, then you can just digest and ligate. If the sticky ends are different from each other, then they will not ligate. You would need to convert the ends to blunt ends either by filling in if 3' overhangs or removing the overhang if 5' overhangs. You can use T4 polymerase for this (it has both polymerase and exonuclease activity) or there are other enzymes that can be used as well. You should in theory only lose the bases from the overhang but occasionally you might lose one or two extra bases.
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