I have transformed dh5alpha cells with pLenti CMV GFP Puro (658-5) constructs (results of Gibson assembly) and while I have colonies on my ampicillin plates, they do not grow in broth (16h at 37). My plates are fresh and the ampicillin is stored at -20, so I am sure that the problem is not the ampicillin. I also used the plates for a different transformation (with pcDNA5.0 constructs) on the same day and those colonies grew in the broth. Someone else had the same problem with her pLenti constructs (alsp from Gibson assembly; she used the same cu vector, but the inserts are different).
Hello,
What concentration of amp are you using for the initial selection (100ug/ml is recommended in this case) ? did you confirm the presence of your construct with colony PCR? if you are using this concentration and confirmed your construct with PCR, the lack of growth in liquids could indicate insert toxicity. If that is the case I recommend switching you ecoli strain to stbl3 or neb stable or neb 10 beta (strains are in order of preference) and growing at 30°C or less when expanding in broth( make sure to run a restriction digest as chances or getting a rearranged vector is quite high in this case). If the problem persists and you are only able to get colonies on solid pates then take a single colony and suspend it in LB (50-100ul) then spread on an LB AMP plate overnight at 30°C then add around 1ml of LB to the grown plate and scrape the plate and transfer the cells suspension to an eppie and continue your mini-prep from there.
Good luck!
Hanna
We are using 150ug/ml because of problems with plates in the past. The construct was confirmed by colony PCR. There are four constucts - all pLenti with varying inserts (2 pairs) so it would be suprising if all four inserts were causing toxicity.
it is often difficult to predict which sequences will cause toxicity. However, if this is truly the case you could do a quick test using already the transformed plate. Try to grow at a lower temperature and see if you get any growth.
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