840:153g:Projects/project25/2012/11/15

Cloning of mreB from Caulobacter crescentus into E. coli

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Experiment 7: Ligation/Transformation and Experiment 8: Plasmid Miniprep Tuesday, November 13th, we assembled transformation reactions with our gene/vector ligation and actually performed the transformation reaction. We plated all reactions on the respective media with selective antibiotic (Ampicillin). The plates incubated overnight and we found the following results. Our 3 ligation tests (L1, L2, L3) all had different insert:vector ratios and all had ample growth. We used these to put together tube cultures (4 replicates of our 3 ligation test reactions). Each tube contained 5mL of LB with 5μL of Amp. One colony was used to inoculate each tube. We had 3 ligation control experiments that tested plasmid without insert and insert without plasmid. There were very few (less than 5) colonies on the latter, the other had no colonies. We believe this to be our plasmid growing, either because it was not properly digested or it somehow was able to be ligated back together - which is highly unlikely. Thursday, November 15, we completed the GeneJET Plasmid Miniprep Kit on our tube cultures in order to isolate our plasmid with the insert (hopefully). We also made glycerol stocks with our remaining tube culture cells. Next class period we plan on verifying the presence of our gene and vector, by gel electrophoresis and/or gene sequencing.