I'm trying to ligate a 2 kb insert into a 3.5kb plasmid, which normally works ok. I transform into top10 competent cells, plate & have colonies the next day. I do colony PCR to check whether my cell have the insert, with primers specific to the ends of the insert and that don't bind to my vector. I only get bands of approximately 600 bp which are all identical. I checked the insert I used in the digest on a gel, there's only the single band at 2kb. Is it likely to be an issue within the bacterial cells or is there another explanation for this? I'm really confused.
This could depend upon what you're trying to ligate. Could any part of the insert be toxic to your TOP10 E. coli (i.e., the part that isn't included in that 600 kb segment, whatever it is)?
The two answers combined could allow you to define your problem- if there is some problem where the insert affects cell growth, and the cells are capable of recombination, then you are selecting for recombination events that alter your insert and prevent toxic effects. You could try using a recombination-deficient strain for cloning, but if this explanation is correct, you will be unlikely to obtain transformants with the offending clone. If that happens, maybe design the clone differently, e.g., think about how to prevent any expression- is this clone designed to be expressed in E. coli?
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