1. How to digest plasmid completely, without even one copy of intact circular plasmid?
2. Is it possible to insert this DNA fragment into the genome of TG1 cells randomly? It looks possible from the paper: Nucleic Acids Research, 1999, Vol. 27, No. 5: 1296–1299. Does anybody have experience?
Rahmat Azhari Kemal
I work with Pichia pastoris, but I hope I can provide some help.
1. After digesting your plasmid, electrophoresis it the purifiy your target band from the agarose. I suggest you do an overnight digestion with lower enzyme concentration (I used 0.25 U per 1 ug DNA for overnight digestion).
2. Yes,it is possible. If your fragment has no homolog with the genome, you might get random integration(s).
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Syed A Ali
To address complete plasmid digestion issue, you can PCR amplify the desired cassette or even the entire plasmid (using whole plasmid PCR) followed by treatment with DpnI, which will chew away all the methylated plasmid DNA used as template. Now, you purify the amplicon and use for transformation. This I do not know. I have engineered recombinant bacterial strains but I used suicide plasmid vectors such as pCVD442 and pWM91.
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