Team:Hawaii/Notebook/2008-08-13

= Things we did today =

Verification of Transformants

 * Grace


 * Colony PCR of transformants and restreaked colonies from yesterday
 * Ran on 2.5% agarose gel at 95V for 100 min.
 * Restreaked:
 * slr1+GFPf colony 5
 * pilA+GFPf colony 4
 * GFPf likely J33207 due to mix up -- see below

Plasmid Prep
Margaret
 * OriV1-4 and aadA(BB) 4 **the only correct transformants from yesterday's ligation/transformation

Ligation
Margaret


 * I corrected my math from yesterday's ligation and re-did the ones that did not ligate correctly

Krystle


 * Ligated using quick ligation buffer and quick ligase:
 * gfpf+B0015(digested by MR)
 * gfpf+B0015(digested by GK)
 * gfp+B0015(digested by MR)
 * B0015(digested by MR) <-- as a control

Transformation
Margaret


 * omega ligation from yesterday
 * rep+B0030, P1+B0015, aadA(pRL1383a) +B0030 from today's ligation

Krystle
 * Transformed using DB3.1
 * Ligation products from today (see above)
 * Notes: Initial incubation on ice only 3 minutes, final incubation with SOC only 1 hour

Gel From Yesterday's Colony PCR
Margaret

Sequencing

 * Grace


 * Checked sequencing results returned from CORE Hawaii
 * Confirmed:
 * B0015
 * B0030
 * B0034 (one bp off)
 * nir
 * slr
 * slr1 = slr2  huh?
 * Other:
 * nir+rbs
 * No rbs present; only nir
 * Huh? We ligated nir INTO rbs
 * I14032+rbs
 * No plac present; only rbs
 * BB-pRL1383a
 * E0040 (GFP) went in successfully, not J33207
 * WTF? Our plasmid preps were/are mislabeled
 * Redo ligation to get blue/white screen?
 * GFP+tt (reverse only)
 * Low quality read; GFP is not present (segment too small)
 * GFPf+tt (reverse only)
 * lacZ fragment present; no tt
 * Huh? We ligated the part INTO tt
 * GFPf+tt and J33207+tt samples switched?
 * J33207+tt
 * Part did not go in; tt only

= Discussion =

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