Team:Calgary/2 July 2009

• Protocol: Used GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH2O.

• Results

Verification digest to verify the presence of restriction sites in the psB1AC3, psB1AK3 vectors as well as in our TOPO TA vector with LuxOD47E.

• Protocol:

5 reactions for psB1AC3 vector, 5 reactions for psB1AK3 vector, 1 reaction for gene of interest (LuxOD47E in pCR2.1-TOPO vector).

BBK Vector Reaction List

Reaction 1- Not1 + React 3 Buffer
Reaction 2- EcoRI + SpeI + React Buffer 4
Reaction 3- XbaI + PstI + React 2 Buffer
Reaction 4- EcoRI + PstI + React 2 Buffer
Reaction 5- XbaI + SpeI + React 4 Buffer

BBK vector tube preparation
8 uL Plasmid (psB1AC3 or psB1AK3)
2 uL appropriate React Buffer
1 uL of each appropriate restrcition enzyme
ddH20 up to 20 uL

TOPO vector tube preparation
10 uL DNA (LuxOD47E in pCR2.1- TOPO vector)
7 uL ddH20
2 uL React 3 Buffer
1 uL NotI restriction enzymes

Left to digest overnight in waterbath at 37°C.

Results:

The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls.

Materials and methods:

These were done in accordance with the procedure outlined on the protocol page.

Results:

These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today.