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

Protocol was followed using ampicillin selection antibiotics for all BioBrick parts and kanamycin for the terminator.

Friday 13th July (9.00)

• Mini-Prepping of pBAD/AraC promoter weak, pBAD/AraC promoter strong and terminator

• Gel Electrophoresis to check fragment sizes of pBAD/AraC promoter weak, pBAD/AraC promoter strong and terminator

o Protocol for Mini-Prep was followed exactly, except:

o For centrifugation of 5mL culture at 3'900rcf for 2 minutes at 21°C prior to the purification protocol.

o Prior to step 4, each Falcon tube containing Lysis and Neutralization Solution in addition to the lysate was transferred to fresh 1.5mL Eppendorf tubes so spinning down would be more effective, conducted at 16'100rcf for 5 minutes at 21°C.

o 850µL of the supernatant was withdrawn (being careful not to disturb the cellular debris) and transferred to a geneJET Miniprep column at step 5.

o The geneJET Miniprep column was then centrifuged at 16'100rcf for 1 minute at 21°C at step 6.

o Centrifugation at step 7, 8 and 9 was conducted at 16'100rcf for 1 minute at 21°C.

o MilliQ H2O (50µL) was added to each Eppendorf and left for a couple of minutes before centrifugation at 16'100rcf for 1 minute at 21°C at step 10.

o The concentration of plasmid DNA obtained in each Eppendorf was measured at the NanoDropping machine.

• 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)