User:University of Washington/5 August 2008

LuxR from pLac
-I0462 part sequence confirmed. Overnight started of that part for glycerol stock.

-R+E sequence appears inconclusive again, alot of n's present.

-Transformation of second R+E ligation plated succesfully on amp plate. Colony selected, and overnight started for sequencing.

-Overnight of DH5a+LacIq started to make electrocompetent.

MG1655Z1
- Restriction digest minipreped DNA of plasmids in MG1655Z1 (expected no plasmids left after cure) with XbaI.

- After 2 hours, ran gel on the digested DNA together with the non-digest minipreped. The result showed that there were still plasmids left in the strain. In the digested DNA lane, there were bands between 5-6 kb, 10 kb and above.

- Streaked MG1655Z1 from glycerol stock made from original strain received on Amp plate to check if there was other unknown plasmids. - Streaked MG1655Z1 from Tsy#2 plate on Amp, Kan, Cam, and Tet plate (All 8 sections) to check what plasmids still existed.

LuxR from AraC and TetR
- Nanodropped the digested P1010(AC): 63.5 ng/ul, 1.02-260/280, 0.27-260/230; and promoter D29: 26.4 ng/ul, 0.81-260/280, 0.33-260/230.

- Ligation of P1010 on pSB1AC3 and promoter 29.
 * Mixed 16.47 ul dH2O + 2 ul T4ligase buffer + 0.32 ul P1010(20 ng) + 0.21 ul D29(~5.5 ng) + 1 ul T4 ligase
 * Incubated 10 ul of the reaction in fridge overnight.
 * Incubated the other 10 ul of the reaction at room temp 30 mins
 * denatured enzyme at 65 degree Celcius 10 mins
 * 20 min on filter paper. (note: mistakenly used Scott's cellulose filter paper, barely got anything left on the paper.)
 * electroporated whatever was left into 45 ul XL1 Blue and plated on Amp.

Lambda Red Recombineering of RP4 (Bryan)
Attempted electroporation of RP1 and recombinant Cam cassette into DY331 with time constants 5.2 & 5.4. Included plasmid-only control to evaluate transformation efficiency, time constant 5.6. Time constants are better than last week's experiments, probably due to improved washing of the target cells and/or a smaller volume of cell/DNA mix (45 uL vs. 75 uL).

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