Team:Exeter/lab book/1gp/wk1

 |  Operon Construction  |  Glycobase  | 

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• Tryptone (10mg) was added to a 1L beaker containing stir bar.

• MilliQ H2O (≈100mL) was then added to the 1L beaker and stirred using a magnetic stirrer.

• Sodium Chloride (NaCl, 10mg) was added to the stirring solution.

• Yeast extract (5g) was then added.

• Finally, agar (15g) was added to the stirring solution.

• The remaining MilliQ H2O (≈900mL) was transferred to the stirring solution and left to fully mix for 5 minutes.

• The LB agar mixture was then transferred to a 1L Duran bottle and thoroughly mixed by inverting the bottle a few times.

• The Duran bottle was then autoclaved for 15 minutes.

Wednesday 11th July (14.30) – BioBrick Extraction and Transformation

• DNA was resuspended in 10µL of MilliQ H2O by pipetting up and down on four selected wells of the five BioBrick plates delivered. These were: pBAD/AraC promoter weak (BBa_K206001), pBAD/AraC promoter strong (BBa_K206000) and a double terminator (BBa-_B0014).

• Punctured wells containing MilliQ H2O and DNA of interest was left for 5 minutes and then transferred to fresh labelled Eppendorf tubes.

• Tubes were centrifuged briefly to spin down any liquid.

• Each BioBrick DNA part (1µL) was pipetted gently into 100µL Top10 E.coli competent cells (Invitrogen), making sure not to mix too rigorously. Three Eppendorf tubes were set up: Tube 1 for 50µL E.coli + 1µL double terminator, Tube 2 for 25 µL E.coli + 1µL pBAD/AraC weak, Tube 3 for 25µL E.coli + 1µL pBAD/AraC strong.

• Competent cells were then incubated on ice for 30 minutes.

• They were then heat-shocked for exactly 30 seconds in a 42oC water bath, making sure not to mix or shake.

• Afterwards, they were quickly placed on ice for 2 minutes.

• Pre-warmed SOC medium (250µL for Tube 1 and 125µL for Tubes 2 and 3) was added and then each Eppendorf tube was secured in a shaking incubator and incubated at 36.8oC for 1 hour at 220rpm.

• Whilst incubating, eight LB agar plates were made-up. Half would be used for spreading 20µL of transformed E.coli competent cells and the other half for 100µL of transformed E.coli competent cells, so that we would be able to pick out enough isolated colonies. Furthermore, six of the eight plates would contain ampicillin and the other two would contain kanamycin (where ampicillin would select all transformed E.coli and kanamycin would select only for the double terminator). Therefore 400µL ampicillin was added to 400mL of LB agar, and 50µL kanamycin was added to 50mL of LB agar to create a 1000-fold dilution.

• After the transformed E.coli competent cells had finished incubating for an hour, 20 or 100µL of the transformants were spread plated onto their respective LB agar plates containing the correct antibiotic.

• Remaining transformation mix was stored at 4oC and the eight inoculated LB agar plates were inverted and placed in an incubator at 37oC overnight.

Thursday 12th July (15.00) – Adding Cultures to Liquid Medium

• Ampicillin, the selection antibiotic, was defrosted.

• Three isolated colonies from the 100µL spread plate that contained our cloned BioBrick parts were picked and inoculated in three separate bottles containing 5mL LB broth (see: Preparation of LB Agar and Broth) via dropping the pipette tip.

• 5µL ampicillin was added to each bottle to make a 1000-fold dilution (since 5mL LB broth used).

• Each bottle was inverted a couple of times, and then put in a 37oC incubator and left 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 21oC.

• 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 21oC.

• 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 21oC.

• 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 21oC.

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

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

• The flow-through was emptied and centrifuged again for 1 minute at 21oC 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, see: Results).

• 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 37oC.

• Loading buffer (4µL) was added to each DNA sample.

• 25µL of each sample DNA was added to different wells, including DNA hyperladder (10µL)