I inserted a 3131 bp fragment to a vector (3661bp) by ECORI and BSP119I enzymes. I performed the colony PCR and saved the positive colonies after cultivation. When I extracted cloned plasmid from bacteria, I observed two bands 3131bp and 800bp but not observed a 3661bp.
Did this happen with all the positive colonies, or did you just extract the plasmid from one of them?
And how large was your undigested vector?
i extract one colony.
the undigested vector (linear) was about 6700 bp at correct length.
I would always recommend extracting plasmids from two colonies, in case something odd happens..
When your undigested vector has the right lenght, I would think something went wrong with the digestion - maybe you picked the wrong enzyme or something? maybe just re-do the digestion just to be sure.
Alright, sorry right now I can't really think of why this would happen. Guess I would just extract new plasmids.
You mean after doing double digest you see two bands - 3131bp and 800bp?
Do two separate restriction reactions - cutting once with EcoRI, and the other one with Bsp ... to see the result. It might happen that you have second place for one of the restrictase generated (it happens ... anyway the DNA polymerase makes mistakes )... you shouldn't see this third 800 bp band. Also separate very well on the agarose gel the restriction reaction, because it might be that the resulting plasmid backbone minus this 800 bp product will co-migrate with your insert.
i performed double digestion at one tube by fast digest thermo enzymes.
Hi there,
I have a few questions for you:
What are the primer sequences for colony PCR? Do they anneal plasmid sequence or insert sequence or both?
Have you run controls for colony PCR with plasmid free clone and with clone containing empty plasmid?
Does PCR work OK with purified plasmid?
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