Team:Calgary/2 June 2009

• Isolated plasmids (pSB1AK3 and pSB1AC3) from overnight cultures prepared by Thane.
• Used GenElute Plasmid Miniprep Kit (Sigma-Aldrich), used as according to the manufacturers instructions.
• Results:

• Set up restriction enzyme digest to ensure that biobrick sites are in the vector. The following are the reactions that were done:

Reaction 1: pSB1AC3 - cut with NotI Reaction 2: pSB1AC3 - cut with EcoRI and SpeI Reaction 3: pSB1AC3 - cut with XbaI and PstI Reaction 4: pSB1AC3 - cut with XbaI and SpeI Reaction 5: pSB1AK3 - cut with NotI Reaction 6: pSB1AK3 - cut with EcorI and SpeI Reaction 7: pSB1AK3 - cut with XbaI and PstI Reaction 8: pSB1AK3 - cut with XbaI and SpeI

• Incubate reactions at 37^oC

• Protocol: Used GenElute Plasmid Miniprep Kit (Sigma-Aldrich), lot # 048k6062, used as according to the manufacturers instructions.

• Results:

• Objective: To verify the presence of restriction sites in the two BBK vectors as well as in our BBK gene in the pCR.2.1-TOPO vector

• 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.