Team:RHIT/Notebook/Journal

Inoculated a colony of E. coli that expressed the alpha mating factor (JW2004-1) in LB broth incubated in shaker at 37°C for 24 hours.

Plated the Blue construct and Red constructs (pRS413A and pRS416A) on LB + Amp and incubated at 37°C for 24 hours.

Inoculated 3 Falcon tubes with 3 ml of LB broth and isolated colonies from the following plates from 6/13: Blue construct, pRS413A, and pRS416A.

Miniprep four overnight cultures of the following for plasmid extraction: E. coli expressing alpha mating factor, the blue construct, and the red constructs pRS413A and pRS416A.

Streak untransformed JW2004-1 E. coli (on M9 minimal media, M9 minimal media +his M9 minimal media +lac, M9 minimal media +his +lac). Observe the growth 24 hours after plating to determine whether or not strain is in fact -his.

Observation: It was expected to see growth on the +his and +his +lac plates, with no growth on the +lac and minimal media plates because of the deletion of the his gene. Strains appear to be growing the same on all medias; however a plating error occurred and LB broth from the inoculated culture of JW2004-1 was streaked on the plates along with the cells, which may create a "false positive" by giving the cells access to histidine. Colonies were picked from the plated JW2004-1 and resuspended in a 5X M9 salt solution prior to plating to wash away LB broth. The resuspensions were plated again on the M9 medias mentioned before plus kanamycin media (to ensure that the strain is Kan resistant as it should be), and the results will be observed tomorrow, 6/26. NEB5α subcloning efficiency cells were also plated with the three colonies.