Team:Exeter/lab book/1gp/wk2

Operon Construction  |  Glycobase

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• DNA was re-suspended in 10µL of MilliQ H2O by pipetting up and down on the well corresponding to an RBS BioBrick (BBa_B0034).

• Punctured wells containing MilliQ H2O and the RBS DNA was left for 5 minutes and then transferred to fresh labelled Eppendorf tube and spun down briefly in a centrifuge.

• The suspended RBS DNA (1µL) was pipetted gently into 25µL of Top10 E.coli competent cells (Invitrogen), making sure not to mix too rigorously.

• 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) was added and then the Eppendorf tube containing transformed E.coli was secured in a shaking incubator and incubated at 36.8oC for 1 hour at 220rpm.

• Whilst incubating, two LB agar plates were made-up, one would be used for spreading 20µL of transformed E.coli competent cells and the other for 100µL of transformed E.coli competent cells, so that we would be able to pick out enough isolated colonies. Therefore 500µL ampicillin was added to 500mL 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 LB(amp) agar plates.

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

Tuesday 17th July (15.00) – Adding Cultures to Liquid Medium

• Ampicillin, the selection antibiotic, was defrosted.

• Three isolated colonies from the 100µL spread plate 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.

Wednesday 18th July (9.00) – Mini-Prepping (Attempt)

• Overnight cultures were transferred into new Falcon tubes and centrifuged at 3901rcf for 10 minutes at 4oC.

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

• It was noticeable that there was no cloudy solution after transferring to new Falcon tubes. Afterwards, when the supernatant was discarded, there was not enough pellet to continue with the mini-prep procedure, and therefore the process of incubating the transformed E.coli overnight in a 37oC incubator was repeated but picking four colonies from the 100µL spread plate instead of three.

Thursday 19th July (9.00) – Mini-Prepping and Gel Electrophoresis

• Overnight cultures were transferred into new Falcon tubes and centrifuged at 3901rpm for 5 minutes at 4oC.

• 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 13000rpm at 22oC.

• 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 13000rpm at 22oC.

• 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 30 seconds at 13000rpm at 22oC.

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

• This was centrifuged again for 1 minute at 13000rpm at 22oC.

• The flow-through was emptied and centrifuged again for 1 minute at 22oC with an empty column.

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

• MilliQ H2O (20µL) 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) by aliquoting 1.5µL of each RBS DNA in triplicate.

• 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 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 37oC.

• 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.