Team:Hawaii/Notebook/2008-10- 9

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Things we did today

Wetlab work

Construction of secretion device (cont.)

Grace
File:100908REdigest.jpg
EtBr stained 4% agarose gel ran at 60V for 3.5 hours. Twenty microliters of RE digested product were loaded into each well.
  • 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 μl ligation reaction to transform DH5α

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.

Construction of plasmid

margaret
File:Rep aadA purification 10 9.jpg
plac/rbs/rep5 (E,S), plac/rbs/rep7 (E,S), plac/rbs/aada (ap32 E,S), plac/rbs/aada ap2 cut with X,P.
  • 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
File:Oriv verification 10 9.jpg
Verification of oriV transformation.
  • 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


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