Team:UCLA/Notebook/Protein Cages/4 August 2015

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Phillip's notes:

Introduction: The gBlocks and primers have all arrived. They will be resuspended, and the initial amplification will be done.

Procedures:

Both primers were resuspended in TE buffer to a total concentration of 100uM. A 10uM stock was then made by diluting in ddH2O. The gblocks were resuspended in 200uL TE buffer to a final concentration of 5ng/uL. A working stock of 1ng/uL was then made using water.

PCR: From NEB, the annealing temperature determined was 65C, so a temperature gradient was ran for each gblock amplification at 61C, 65C, and 69C.

A master mix was created for 6 reactions with the following reagents per reaction. 5x Q5 buffer, 5uL 2mM dNTP, 2.5uL 10uM PCquad prime 3.0 primer F, 1.25uL 10uM PCquad prime 3.0 primer R, 1.25uL 0.25uL Q5 polymerase 0.25uL of 1ng/uL template Water up to 25uL.

The master mix was made without the template, and 24uL were aliquotted to 6 PCR tubes. The template was added individually to each PCR tube.

Thermocycle conditions: Initial denaturation: 98C, 30s 25 cycles of the following: 98C, 10s; annealing temp, 15sec; 72C 30s Final extension: 72C 5min Hold: 4C

Gel electrophoresis: To each PCR tube, 5uL of loading buffer was added. For the PCquad prime 3.0, 28uL of solution was loaded to the gel. For the PCquad mutant 10, 20uL of solution was loaded since the gel was thinner. 10uL of ladder was used for both. Electrophoresis ran at 100V for 20 minutes.

Results: The PCquad prime 3.0 seems to have amplified fine. 66C showed the brightest band for the product size ~1.5kb. The PCquad mutant 10 shows one clean band, however, it is at a size ~400bp greater than expected.

Media:2015-08-04 PCR PCquad mutant 10 amplification 61C, 65C, 69C From left to right for both: gene ruler ladder, 61C, 65C, 69C

The bands from PCquad prime 3.0 were cut out and put in a tube in the -20C.

Conclusions: It appears as if the new primers and construct work quite cleanly. The next step would be to either rerun the reaction in larger scale (after consultation) or to gel purify the excised band. The PSB1C3 vector will need to be obtained, and then digestion and transformation can be done.