Week 1 Lab Summary (5/7/18-5/14/18)
This week my team and I setup a digest for pGADT7-TRIM5α, from a miniprep done by J. Graff previously in the semester. The digest from this sample was lost due to the addition of a ladder being inserted into the sample. The miniprep sample used for this sample ran out, so we started a new culture of pGADT7 and miniprepped that culture to obtain more plasmid to run a digest. Concentration of the miniprep was low so we are going to start and miniprep another culture of pGADT7-TRIM5a. Another solution is to use the low concentration as template for a transformation into E. coli and to do a miniprep on yielded colonies to obtain a better concentration.
PCR amplification of TRIM8 and TRIM13 was unsuccessful with Human cDNA as the template DNA. It is believed that the incorrect concentration of Template DNAwas used so we nanodropped the sample and redid calculations for the correct amount of cDNA to be used in the Thermocycler PCR protocol.
pTWIST-TRIM5α arrived so we transformed the plasmid into E. coli and then picked an isolated colony and grew a culture in LB broth +amp. The culture was miniprepped and nanodropped. That sample was then used in a digest with EcoRI and NotI and a picture was taken for confirmation that TRIM5α was properly extracted from the pTWIST plasmid.