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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

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

Team:Hawaii/Notebook/2008-09-25