You perform a ligation with NcoI/NotI-digested pET-41a vector and the egfp insert from pEGFP-N1 (as in the lab). You then transform the ligation mix into E. coli and plate on LB medium containing kanamycin. Consider the following unexpected outcomes, and suggest controls: a. Nothing grows. What controls might you design to determine whether the E. coli cells are viable (alive) versus whether the E. coli is competent

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Biology

Gwendolyn Diaz

4 November, 14:05

You perform a ligation with NcoI/NotI-digested pET-41a vector and the egfp insert from pEGFP-N1 (as in the lab). You then transform the ligation mix into E. coli and plate on LB medium containing kanamycin. Consider the following unexpected outcomes, and suggest controls: a. Nothing grows. What controls might you design to determine whether the E. coli cells are viable (alive) versus whether the E. coli is competent

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Melanie · Answer 1

You can do two controls:

1) Check if the cells are viable by plating the transformed E. coli on LB medium with no antibiotics. If the cells are alive, a lawn will be seen, as there is no selection pressure. If the cells are dead, no colonies will be seen.

2) Check if the cells are competent by transforming them with a control plasmid. This is another plasmid, either commercial or already used in the lab, that contains the gene for resistance to kanamycin, and is known to work. If this positive control is used to transform the cells and still no growth is seen in the LB plates, then the E. coli are probably not competent.