Field of Science

Today's experiment

Today I'm planning to do the first of what I hope will be a long series of natural transformation experiments in E. coli.  The research associate has already done a number of these, but I didn't trust her positive results because they happened in the supposedly recA- strain DH5alpha as well as in another recA+ strain.  But now I've found that our DH5alpha stocks have the UV-sensitivity typical of recA+ strains I'm more optimistic that the transformations really are working.

What strains will I test?  The two strains that she has previously tested, plus a control strain that doesn't carry the plasmid we use to induce the chromosomal competence genes.  (I could not bother with the DH5alpha strain, but I think its unexpected phenotype may provide useful information.)

What DNA will I use?  The RA has made a stock of DNA from a strain carrying a kanamycin-resistance insertion in the crp gene.  She uses it at 2 micrograms/ml.  She's now making a better strain whose DNA we'll use for future experiments; it carries this insertion and easily-selectable wild-type alleles of other genes.

How will I prepare the cells?  I've grown the two plasmid-carrying cultures overnight from single colonies (over two nights really as I inoculated them on Friday night).  I'll dilute these 1/100 in LB and grow them to OD=0.2  The other strain I'll need to inoculate directly from a single colony this morning.  When the cells are at OD=0.2 I'll add IPTG and then the DNA.  After 2 hours I'll plate the cells with and without kanamycin.  I'll use a wide range of dilutions on the kanamycin plates, because the RA has found 'bald-spot' problems when dense cultures are plated directly.  I'll include a negative control with no DNA, and another with DNaseI added at the same time as the DNA.

What do I need to do to prepare?  Pour lots of LB plates, especially kanamycin ones.

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