Hi all,
I made a double digestion of a plasmid (1 ug) using 20U of each EagI-HF and NcoI-HF REs and 1x rCutSmart buffer in a 50 ul final volume. The reaction was incubated at 37 ˚C overnight. On the next day, I checked the double digestion (2 bands seen in the gel) and purified the heavier band from the gel (the linearized plasmid). To confirm the complete double digestion, I then used 1 ul of the purified linearized plasmid in a PCR with M13 primers (0.2 uM end-concentration). No amplification was expected since EagI-HF and NcoI-HF should have removed the sequence between the M13 biding sites of my plasmid. However, the PCR produced a strong band of the exact expected size for the amplification of the sequence between M13 sites.
Does that mean that the double digestion didn’t work at all? Maybe there is a PCR bias favoring the small amount of intact (non-digested) plasmid? In that case, is PCR with M13 primers a valid way to test for complete double digestion of a plasmid?
Thanks in advance,
Dani.
I'm not sure how useful this will be for you. Even if you had 99.9999% digestion, even a few molecules of undigested plasmid might still give you a signal with PCR. The question is whether you really need to worry about it? Alternately you could just take an alilquot and use for transformation without ligation and that will also give you an idea of the functional background of undigested plasmid.
Thank you Michael, I agree with you. None reaction will be 100%. Transform bacteria with an open plasmid might be a better control indeed.
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