I am doing a ligation with a 3.8kb vector and inserts are 672 bp. The vector is ampicillin resistant. I got every time sharp band (sharp band linearazed plasmid DNA and insert band) on gel from insert amplification to Restriction digestion step. After that, I proceed the Transformation step, I transformed into lab made competent cells DH5α (Ecoli cells).after transformation, I got equal no. of colonies on control and experimental ampicillin resistant plates. Any suggestion as to why this happened?
If you wait long enough, satellite colonies that don't harbour the plasmid will grow on ampicillin plates, owing to the destruction of ampicillin by the beta-lactamase produced by the resistant clones. For this reason, I prefer to use carbenicillin as a selecting agent, particularly if I plan to wait before collecting transformants (e;g. by letting the colonies grow at room temperature on the bench over the week-end).
What restriction enzyme(s) do you use?
Do you perform a vector dephosphorylation step to prevent re-ligation?
Hello Christoph Paone,
I used restriction enzyme EcoR1 and Not1. for dephosphorylation ,i used CIP.
Hi,
in your case the problem isn't vector dephosphorylation because you haven't any colonies on the control!
I suggest you to increase the quantity of digested vector.
Are you sure that the insert is correctly digested?
Which ratio do you use for the ligation reaction?
Hi,
It can happen for several reasons. I will suggest you to make fresh competent cells before the experiment. I will also suggest you to use higher concentration of digested plasmid as well as you can also increase concentration of antibiotic in the plates. I generally use .2% of ampiciline having concentration 100 microgram/ml you can increase ampicillin concentration. Also carefully mix competent cells with your digested plasmid by pipetting before heat shock. All the best.
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