hi, we are working on the puc57 plasmid that our construct designed with multiple RE sites and synthesized and cloned in it. we use fast digest re enzymes (thermofisher) to separate the parts of our construct for sub-cloning.
we have some issues with these enzymes that didn't work well on the electrophoresis with TAE buffer and the expected parts of the gene after restriction, are invisible, we did simple digest and double digest with one and two enzymes. what do you think is the problem?
thank you
have you run pcr with a primer in the vector to a primer in the construct? This is necessary as primers only in the construct may amplify fron unincorporated construct left over from the ligation reaction and you may not have any insert for the enzymes to work on. Is the construct big enough so that if you use a single cutting enzyme it will be possible to see dna with and without insert just to show that you have an insert
That is good suggestion by Paul Rutland but in addition you could test your plasmid as to whether it digests with each enzyme alone. Run uncut plasmid, cut with enzyme 1, cut with enzyme 2, and cut with both and see how the gel looks. It may be that one enzyme does not cut well, or it may be that there is something wrong with the plasmid. Be sure the linearized size is as expected.
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