Team:Calgary/12 June 2009

Looked at the Optimization Toolbox:

• Had a team meeting where each subproject presented their recent work. Lab group talked about verification of genes of interest in TOPO vectors, BBK ampliciation PCR and cloning into Biobrick vectors (psB1AC3 and psB1AK3).

Procedure for RD:

oth the vector and the insert need to have their own separate tube, at least in the beginning. This is important because it allows for clean addition new parts to a the circut

In the Insert Tube...

-600ng of DNA (To figure out the volume, the calculation is 600 / concentration of plasmid. This gives you volume in μL). -Water, so that the volume of DNA and water in the tube is 35 μL -4 μL of React 1 Buffer -0.5 μL of EcoR1 -0.5 μL of Spe1

In the vector Tube...

-250ng of DNA (To figure out the volume, the calculation is 250 / concentration of plasmid. This gives you volume in μL). -Water, so that the volume of DNA and water in the tube is 35 μL -4 μL of React 2 Buffer -0.5 μL of EcoR1 -0.5 μL of Xba1

Put both tubes into the 37°C water bath for one hour. After, place them into the 65°C heating block for 10 minutes. This destroys any enzymes in the tube (which is ok, because by now they’ve done all they need to). Take the insert out, and put it in a -20°C freezer.

Protocol: Please refer to the protocol page. Concentrations were measured after the product was purified.

Protocol:

This is the restriction digest component of the construction protocol, which we describe on our protocol page. We attempted the construction with 2 different sets of enzymes: 6 tubes with cuts at XbaI and PstI , and 6 tubes with cuts at EcoRI and PstI. Both were prepared in REact 2 buffer. Once prepared, the tubes were left in the water bath at 37 degrees C for 6.5 hours; heatshocked at 65 degrees C on a heating block to deactivate the restriction enzymes in the tube; and placed in the -20 degrees C freezer.