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#1
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| I have some problem by using pcDNA 3.1/Myc-His(+) B. I am trying to cut Hind3 and Kpn1cutting sites in order to insert my favorite gene. Since I have tried several times and still can not get my favorite gene inserted, I was wondering is it possible to cut pcDNA 3.1/Myc-His(+)B by Hind3 and Kpn1 at the same time? Would the two restriction enzymes compete with each other since their cutting sits are near by? |
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#2
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| In article <[Only registered users see links. ]>, [Only registered users see links. ] wrote: Their sites are right next to each other, which might indeed be a problem. It's not competition but the fact that many restriction enzymes need extra dsDNA on both ends of their recognition sequence. KpnI is is supposed to cut well (50-100% activity) with just one base beyond recognition site. HindIII, however, is more demanding: no cut at all with one base beyond its recognition, 0-20% with two bases, 50-100% for three (according to Fermentas table). So, apart from using different enzyme pair or doing restriction enzyme-independent cloning, your choices are: 1) Do HindIII digestion in NEB buffer 2, then, after verifying that it is complete, throw in as much KpnI as reaction conditions afford and hope for the best. 2) Clone something/anything into HindIII site (obviously, orientation does not matter in this case; any fragment or 20 bp annealed oligos), then the double cut will be efficient. (And, in the case of large fragment inserted, double cut will be eay to monitor/verify). DK |
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| 31 or mychis , cutting , pcdna , problems |
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