I am growing E. coli transformed with the plasmid pUC19 with an insert of an enzyme in rat brain between the mentioned cut sites (EcoO109i and HindIII). I have isolated and created the recombinant molecule with custom gene synthesis so that the gene encoding for the enzyme easily ligates into the plasmid. It is after the lac promoter and lac operator, so will I need lactose in the growth media for the enzyme to be made?

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    $\begingroup$ Why would you cut out the entire lacZ operon instead of using the MCS? Without any of the machinery expressed from the operon, who knows how well expression of your target gene will work? $\endgroup$ – MattDMo Jun 3 '16 at 11:35
  • $\begingroup$ @MattDMo lacZ is a single gene, not an operon. The only negative effect I can think of (assuming there are no issues from removing the fragment between lacZ and ampR) is the inability to do blue-white screening with the plasmid. $\endgroup$ – March Ho Jul 3 '16 at 15:00

I would rather use the synthetic analogon IPTG for induction of genes behind a lac promoter, as they are not getting degraded (or at least much slower than lactose) and thus give a much stronger induction signal.

You will need to test the optimal IPTG concentration, but as a starting point I would use and endconcentration of 1mM. If this is not optimal (to much unsoluble protein or not enough), you can change the concentration in either direction. To start, use an overnight culture to inocculate a fresh culture in the morning and follow up the OD600 until it reaches 0.6-0.8 before adding the IPTG.


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