I have tried three different vectors including pHis, pQE30, and pET47b(+) and BL21 DE3 strain of E.coli. Clones are PCR positive but on digestion I am getting smears.
If you don't use an endA- strain to make plasmid, any contaminating endonuclease that co-purifies with the plasmid starts to act as soon as you incubate it in a buffer containing Mg. It is usually best to clone into a good restriction- host eg. XL-1 and check construct for insert/mutations before transforming into an expression host.
I'm assuming you are screening clones by colony PCR and that the internal primers are for your recombinant protein. Therefore all things being well if you are digesting your PCR product there should not be any contaminating Dnases. Have you tried fresh endonucleases as this is the probable source of the problem.
I have cloned the gene forst in pGEMTeasy and then in respective vectors. I tried both coloby pcr as well as plasmid pcr, all is well but getting smears on digestion, tried with fresh enzymes but result is same.
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