Inoculated 5mL of LB + Amp (100ug/mL) in 50mL conicals with one of the following Withering Syndrome clones frozen stocks (located in -80C box called “WS RLP Plasmids”):
- PWC8 WFS-RLP pCRII (from 4/13/01)
- p16RK3 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
- p16RK7 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
- p18RK7 WFS-RLP rDNA pCR2.1 (from 4/15/01)
Incubated O/N, 37C, 200RPM. Will isolate plasmid DNA tomorrow.
We identified a usable clone for the RLP qPCR assay that contains the entire GenBank sequence (AF133090), instead of the partial (and incorrect) clone that Lisa and I had used previously (see 20120323); leading to a month’s worth of qPCRs that failed to produce the expected, and desired, results. This clone is vector pCR2.1 (Invitrogen) containing the full GenBank sequence, AF133090).
Since the p16RK3-C clone failed to grow yesterday, decided to make cultures from all three frozen stocks of p16RK3 to ensure that at least one will grow.
Three 5mL O/N cultures in LB+Amp (100ug/mL) were inoculated directly from the frozen stocks and grown at 37C on a rocker in a 15mL conical tube.
All three cultures failed to grow. Left cultures in incubator on rocker. Will make some LB and LB+Amp plates and try streaking out all three frozen stocks. Also will try growing the frozen stocks in liquid LB (no Amp) O/N in hopes of getting a culture for mini prep.