Team:WashU/Week8
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- | This morning, we miniprepped a culture of PC42, our ligation of plasmid PSL2131 and construct CS42S. Then, we ran a gel of double and single digests of PC42, cut with X and P and just P, along with an uncut PC42 as a control. [ | + | This morning, we miniprepped a culture of PC42, our ligation of plasmid PSL2131 and construct CS42S. Then, we ran a gel of double and single digests of PC42, cut with X and P and just P, along with an uncut PC42 as a control. (2x refers to ligation cut with both X and P, 1x indicates ligation cut with P, and 0x signifies uncut PC42) |
+ | <div align="center">https://lh5.googleusercontent.com/--ulNrdD-1xs/UAhk8NfQmeI/AAAAAAAAAV0/BVhDt7rnPAY/s800/pc42digest%25207-18.jpg</div><br> | ||
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+ | In addition, we took the cultures of the double digest from Monday, miniprepped them, nanodropped, and then digested them with EcoRI and PstI. The digestions were then run on a gel. [PICTURE BELOW] | ||
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Finally, we conducted an experiment to see if the copy number of a plasmid could be regulated by varying the antibiotic concentration. To do this we grew our <i>E. coli</i> which was transformed with both the zeaxanthin producing construct and our Cs42s construct with varying amounts of chloramphenicol while holding ampicilin concentration constant. We then analyzed the recovered plasmid by digesting with XbaI and PstI and comparing the intensity of the digested plasmids on ethidium bromide stained agarose gel electrophoresis. The results below demonstrated that above a critical concentration needed to select for the double transformed cultures, copy number is unaffected by antibiotic concentration. In the picture below, the wells, from left to right, have 5 microliters of DNA followed by 10 microliters of DNA per each concentration of chloramphenicol, which is labelled on the graph as 0, 25, 50, 75 and 100 mM. | Finally, we conducted an experiment to see if the copy number of a plasmid could be regulated by varying the antibiotic concentration. To do this we grew our <i>E. coli</i> which was transformed with both the zeaxanthin producing construct and our Cs42s construct with varying amounts of chloramphenicol while holding ampicilin concentration constant. We then analyzed the recovered plasmid by digesting with XbaI and PstI and comparing the intensity of the digested plasmids on ethidium bromide stained agarose gel electrophoresis. The results below demonstrated that above a critical concentration needed to select for the double transformed cultures, copy number is unaffected by antibiotic concentration. In the picture below, the wells, from left to right, have 5 microliters of DNA followed by 10 microliters of DNA per each concentration of chloramphenicol, which is labelled on the graph as 0, 25, 50, 75 and 100 mM. |
Revision as of 19:55, 19 July 2012