Double digested plasmid and insert using Sac I and Kpn I. Purified the products after gel elctrophoresis using the qiagen kit. Ligated the products using standard T4 ligase (brand new) over night at 4 deg.
Transformed DH5alpha cells using electroporation, using 1 microL of the ligation. Here, I normally precipitate out the DNA before electroporation, but decided to risk it... there was no arcing. I did not dilute the ligations. I have no colonies apart from the positive control, which was 20 ng of uncut, empty vector.
When plating out the cells, I pelleted them and resuspended them in 200 microL of SOC before plating out about 100 microL of the denser suspension. It doesn't mention this on any protocol but my supervisor told me to do so when I first did some cloning (which did work initially).
The constructs are all good, the restriction enzymes and the ligase are all active as I have tested them. What else could it be?
I realise this kind of question gets asked all the time. Thank you very much for any responses.
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Transformation problems again...
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