Team:Hawaii/Notebook/2008-08-31

= Things we did today =

Construction of p+r (cont.) and re-replacement of BB-pRL1833a MCS

 * Grace


 * Ran RE digests from last night on gel
 * plac+rbs band not visible; will ligate using plac and rbs parts (instead of plac+rbs part)
 * Image to right has been edited w/ Photoshop. Faint smear for plac+rbs was not visible before.
 * Extracted bands from gel
 * Ligated:
 * plac and rbs (B0034) and C0012 vector
 * J33207 and BB-pRL1383a
 * Transformed into DH5&alpha; cells
 * Plated BB-pRL1383a+J33207 on selective media with and without IPTG to verify if IPTG is necessary for blue/white screening in this strain

Inoculated TB+sp100 with BB-pRL1383a

 * Grace

Construction of Broad-Host-Range Plasmid Parts

 * Margaret


 * gel of yesterday's re-digest, lane 3 was cleaned using gel purification spin columns and stored in -20&deg;C. Lanes 7-10 were extracted and cleaned as well.
 * Ligation: using T4 ligase, 10X buffer (from the same lot as the enzyme, it was aliquoted today). 2 hour incubation at room temperature. The reaction was placed in 4&deg;C in case transformation does not yield colonies.


 * Transformation: of ligation products from today into DH5-alpha cells. Everything plated on Amp100 LB plates, pSMC121 used as positive control (plated on SmSp).


 * Plasmid Prep: stored in -20&deg;C in 30ul TE buffer
 * Culture: 5mL Terrific Broth + amp100
 * plac+B0030 (1:6):3, 4, 7
 * plac+B0030 :3
 * plac+B0034 (1:6):1
 * plac+B0034 (1:3):3

= Discussion =

= Quote of the Day = "History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson"