Field of Science

Follow-up frustrations

I'm trying to do the follow-up experiment to the previous unsuccessful attempt to transform E. coli with chromosomal DNA. This 'follow-up' is really a 'fall-back', as I'm now trying to replicate the RA's previous apparently successful transformations. I'm growing the two strains she's transformed previously (derivatives of BW25113 and DH5alpha, each containing a low-copy sxy expression plasmid), and will transform them with the two DNAs she's used successfully, one from RR902, an old E. coli strain containing a Tn10 insertion in purE*, and the other from RR1314, a BW25113 derivative containing a KanR cassette in crp. Both genotypes have been confirmed; RR902 grows fine on minimal supplemented with the purine inosine but not at all without inosine, and PCR shows the size of the crp fragment in RR1314 to be that predicted by the disruption.

Because she's found plating anomalies depending on cell density, I'm planning to plate a wide range of dilutions, and I'm also going to use two kanamycin concentrations - the 10 µg/ml she's already used and also 20 µg/ml.

BUT... one of the recipient strains (RR3013, the DH5alpha derivative) refuses to grow in LB with 20 µg/ml chloramphenicol (the selection for the sxy plasmid), although the other strain, which carries the same plasmid, is growing just fine. Both were inoculated at the same time, each from a plump single colony on a LB Cm10 plate. And when I tried to look at the non-growing culture under our very expensive microscope, I discovered that the high-power lens has somehow become all crudded up, and that lens cleaning solution doesn't help.

So I guess I'll just do the transformations into the other strain (RR3015). Update: my reinoculation of RR3013 crept up to the appropriate density so I included it too.

* Our strain list says this strain is also called NK6051 (constructed by Nancy Kleckner), and the E. coli Genetic Stock Center says NK6051 has its Tn10 insertion in purK, not purE. That's fine, purK is the gene next door to purE, so the original mapping was probably an error.

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