Brown: Team Resistance/22 October 2008

22 October 2008

Ligation

pVJ4 lysis cassette and S105 lysis cassette into standard biobrick vector plasmid pSB1AK3 at EcoRI and SpeI

• 15 ul mixes:
• 12.37ul diH20
• 1.5ul T4 10x buffer
• 2.08ul pVJ4 OR

2.62ul S105

• 0.53uL vector
• 0.5ul T2 ligase

Transformation

• Used 50ul competent cells (invitrogen)
• skipped the SOC step (immediately plated) since SOC step not necessary for amp resistant plasmid
• Put in incubator at 10:30am

Conductance test

Measuring conductance change and optical density change in parallel

Set up two cultures of the pVJ4 plasmid

1. control (no ara*)
2. test (480ul ara*)

Because the cells grew during the experiment into stationary phase, the lysis seems to take much longer than usual.

We measured conductivity and optical density of solutions every 15 minutes. Initially we had tried leaving the conductivity probe in the solution of cells, allowing the probe to take continuous readings as the cells lysed in solution. However, the probe does not seem suited to this function. It is recommended that probes be cleaned between every reading, so we are rinsing the probes with milli-q water and drying them with compressed gas between every reading.

Conductivity 1: solution with ara Conductivity 2: solution without ara

• We created a 50% filter sterilized arabinose solution. 480ul is 2% arabinose in of bacterial solution.