Team:Hawaii/Notebook/2008-09-25

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Things we did today

Wetlab work

Verification of Ligation

Margaret
  • The plac/rbs+rep and oriTr(BB) transformations resulted in 0 colonies with the correct insert, so the remainder of the ligation reaction from 9/16 was digested with XbaI and run on a gel.

Sequencing & Colony PCR

Margaret
  • PCR
  • oriV colonies (3,4,6,8,9,10,11)
  • aadA(BB) colonies (4,5,7,8)
  • aadA(pRL) colonies (1,2)
  • oriT + J33207 (1,2,4)
  • oriT + J33207(de-phosphorylated) colony 1
  • 5 additional colonies of oriTr transformation were PCR'ed


Construction of secretion device and redo of BB-pRL1383a (cont.)

Grace
EtBr stained 2% agarose gels ran at 60V for 1.5 hours and 2 hours, respectively. Thirty microliters of RE digested products were loaded into each well.
  • Ran RE digests from last night on gel
  • nrsg8 and nrg4 did not cut. Implies insert wrong?
  • Extracted bands from gel
  • Ligated:
  • 3A using pSB1A3:
  • nrg7 + tt
  • rgt1 + nir
  • rgt1 + plac
  • rgt2 + nir
  • rgt2 + plac
  • pSB1A3 (control)
  • pRL1383a + J33207
  • pRL1383a to self (control)
  • Transformed into DH5α using 5 μl ligation reaction; also transformed J33207 and pRL1383a plasmids as control)

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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