Team:Hawaii/Notebook/2008-10- 9

= Things we did today =

Construction of secretion device (cont.)

 * Grace


 * Ran RE digests on gel
 * Extracted bands from gel
 * Ligated:
 * J33207 and pRL1383a
 * nir and rbs+GFPf+tt #1 and pSB1A3
 * plac and rbs+GFPf+tt #1 and pSB1A3
 * nir+rbs and slr1+GFPf and pSB1A3
 * nir+rbs and pilA+GFPf+tt and pSB1A3
 * Used 5 &mu;l ligation reaction to transform DH5&alpha;

Sequencing

 * Grace


 * Prepared and sent samples in for sequencing:
 * pilA+GFPf+tt #21 (KS)
 * nir+rbs+slr1+GFPf #15 (10/2 transformation)
 * slr1+GFPf+tt (10/2 transformation)
 * nir+rbs+slr1+GFPf #6 (10/8 transformation)
 * plac+rbs+pilA+GFPf+tt #5, 7, 11 (10/8 transformation)

Triparental conjugtion

 * Grace


 * BBpRL #1, 18 cultures did not grow
 * PCR of colonies (on Xgal plate) showed no insert present (?!?)
 * PCR of colonies from non-Xgal plate) showed no insert present (?!?)
 * Faint band for colonies #1, 13 at 400bp ???
 * Restreaked original colonies #1, 3, 13, 18 on freshly made LB+sp100+sm100
 * Original colonies scraped w/ loop. Hopefully there's something there.

Made 5%LB+BG-11 and BG-11+sp2.5+sm2.5 plates

 * Grace and Krystle

Construction of plasmid

 * margaret


 * Gel purification of re-digest
 * plac/rbs/aada (ap32 was cut with E,S), ap2 did not cut correctly
 * plac/rbs/rep (7 was cut with E,S), rep5 did not cut correctly
 * Ligation, transformation (in DH5 alpha):
 * oriT + plac/rbs/aada + pSB1A3 (dephosphorylated)
 * oriV + plac/rbs/rep + pSB1A3 (dephosphorylated)
 * pSB1A3 (dephosphorylated) + self
 * (+) control for transformation- pUC18
 * (-) control for transformation- no plasmid

OriV back-ups

 * margaret


 * two colonies were of correct size from yesterday's transformation, so i did a plasmid prep of them and I will store them in -20 C in TE

= Discussion =

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