qPCR – RLP Plasmid Curve Comparison: Lisa vs. Sam

Performed qPCR on dilutions from the p16RK3-A NcoI linearized plasmid prep from 20120419. Lisa used the stock p16RK3-A NcoI linearized plasmid prep from 20120412 to make her own set of dilutions. Both of us will run these samples on the same plate and will use the Stratagene MX3005P, NOT the BioRad CFX96.

The rational is that this plasmid prep should contain the correct insert and we will see if the problems I’ve been experiencing with qPCR of all these RLP curves is due to something I’m doing or if the lab has just been cursed. Additionally, performing the qPCRs on the Stratagene will also rule out any effect(s) that the BioRad machine might be having on the data (although, the BioRad being screwed up is very unlikely).

Master mix calcs for my reactions are here. All samples were run in duplicate. Plate layout, cycling params, etc.

Results:

qPCR Data File (MX Pro; Requires MX Pro v 4.10)

Bacteria Cultures – p16RK3-A, B, & C Clones from 5/2004

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.

Results:

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.