Team:Exeter/lab book/novpol/wk2

Operon Construction  |  Glycobase

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**Monday 16/07/12**

Performed the BioBrick extraction of RBS BioBrick (BBa_B0034) and TetR promoter. Subsequently Transformed the BioBrick onto 50ul of competent cells and plated onto agar containing ampicillin.

**Tuesday 17/07/12**

Cultures containing the RBS and TetR promoter were transferredto liquid medium containing ampicillin.

**Wednesday 18/07/12**

RBS mini-prep attempt seemed to not pellet so transferred new colonies into liquid broth again containing ampicillin.

**Thursday 19/07/12**

Mini-prep and gel electrophoresis of the TetR promoter and RBS

To verify BioBrick parts were cloned successfully, gel electrophoresis was used. Agarose was made (1x 50mL TAE buffer, 0.5g agarose and then microwaved until melted). Ethidium bromide (EtBr, 1µL) was added to the agarose gel, and then mixed. The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted. The BioBrick parts were removed from the circular plasmids by transferring each cloned plasmid containing different BioBrick parts to different Eppendorfs containing the Master Mix. This consisted of: 500ng/µL RBS DNA, 1µL EcoR1-HF, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and enough MilliQ H2O to make a 50µL total volume. The resulting Master Mix was multiplied by four to compensate for determining the sizes of the four BioBrick parts. The DNA-Master Mix solution was left to incubate for 10 minutes at 37°C. Loading buffer (4µL) was added to each DNA sample. 25µL of each sample DNA was added to different wells, including DNA hyperladder (10µL). Gel electrophoresis was then run for approximately 40 minutes at 150V.