Team:Exeter/lab book/novpol/wk1

Operon Construction  |  Glycobase

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**Tuesday 10/07/12**

Today we had our laboratory induction, we were shown where all the equipment was and given a demonstration.

LB agar and Broth were then prepared.

**Wednesday 11/07/12**

Today we performed the BioBrick extraction of: pBAD/AraC promoter weak (BBa_K206001), pBAD/AraC promoter strong (BBa_K206000) and a double terminator (BBa-_B0014).

Subsequently transformed the BioBricks onto 100ul of competent cells and plated onto agar containing ampicillin.

**Thursday 12/07/12**

The cultures containing the pBAD/AraC promoter weak, pBAD/AraC promoter strong and the double terminator were transferred to liquid medium containing ampicillin from the plates. Three separate colonies were selected for each BioBrick to produce replicas.

**Friday 13/07/12**

The pBAD/AraC promoter weak, pBAD/AraC promoter strong and the double terminator genes were mini-prepped. The concentration of plasmid DNA obtained in each Eppendorf was measured at the Nanodropping machine (in ng/µL, see: Results). The genes were then separated on gel electrophoresis.

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 in the fume cupboard, 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 DNA of interest, 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 selected. 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 20 minutes at 150V.