Team:Exeter/lab book/1gp/wk1

Operon Construction  |  Glycobase

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Wednesday 11th July (14.30)

• BioBrick extraction of pBAD/AraC promoter weak (BBa_K206001), pBAD/AraC promoter strong (BBa_K206000) and a (double) terminator (BBa_B0014)

• Transformation of pBAD/AraC promoter weak, pBAD/AraC promoter strong and terminator

2 x 25µL and 1 x5 0µL of One Shot® competent cells were used instead of 3 x 50µL for all transformations.

Thursday 12th July (15.00)

• Adding cultures with pBAD/AraC promoter weak, pBAD/AraC promoter strong and terminator into liquid medium overnight.

Friday 13th July (9.00) – Mini-Prepping and Gel Electrophoresis

• Overnight cultures were transferred into new Falcon tubes and centrifuged at 3900rcf for 2 minutes at 21°C.

• The supernatant was discarded (being careful not to disturb the pellet).

• The pellets were re-supended in Resuspension Buffer (250µL) by pipetting the solution up and down.

• Lysis solution (250µL) was added and immediately Neutralisation Buffer (350µL).

• Each Falcon tube was then centrifuged for 5 minutes at 16100rcf at 21°C.

• 850µL of the supernatant was withdrawn (being careful not to disturb the cellular debris) and transferred to a geneJET Miniprep column.

• The geneJET Miniprep column was then centrifuged for 1 minute at 16100rcf at 21°C.

• The flow-through was discarded (as this contained the sugars, metabolites etc.) and then 500µL of Wash solution was added to the geneJET Miniprep column.

• This was centrifuged again for 1 minute at 16100rcf at 21°C.

• Any flow-through was discarded and washed again with extra Wash solution.

• This was centrifuged again for 1 minute at 16100rcf at 21°C.

• The flow-through was emptied and centrifuged again for 1 minute at 21°C with an empty column.

• The supernatant obtained was transferred to clean, labelled Eppendorf’s.

• MilliQ H2O (50µL) was added to each Eppendorf and left for a couple of minutes.

• The concentration of plasmid DNA obtained in each Eppendorf was measured at the Nanodropping machine (in ng/µL).

• 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 Eppendorf’s 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)