Team:Exeter/lab book/novpol/wk1

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ExiGEM2012 Lab Book NovPol wk1


\| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid \| \| Operon Construction \| Glycobase \|
9th - 13th July - 16th - 20th July - 23rd - 27th July - 30th July - 3rd August - 6th - 10th August - 13th - 24th August - 27th - 31st August
	Showcasing Polysaccharide Production: 9th - 13th July 2012 Tuesday - 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. Tryptone (10mg) was added to a 1L beaker containing a stir bar. MilliQ water (≈100mL) was then added to the 1L beaker and stirred using a magnetic stirrer. Sodium Chloride (10mg) was added to the stirring solution. Yeast extract (5g) was then added and finally agar (15g) was added to the stirring solution. The remaining MilliQ water (≈900mL) was transferred to the stirring solution and left to mix for five minutes. The LB agar mixture was then transferred to a 1L Duran bottle and then autoclaved. Wednesday - Today we performed the Biobrick extraction and Transformation. DNA was re-suspended in 10µL of MilliQ water into four selected wells of the Biobrick plates. These were: pBAD/AraC promoter weak (BBa_K206001), pBAD/AraC promoter strong (BBa_K206000) and a double terminator (BBa-_B0014). These were left for five minutes and then transferred to labelled Eppendorf tubes. The tubes were centrifuged briefly to spin down any liquid. Each BioBrick DNA part (1µL) was pipetted gently into 100µL of 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 placed on ice for 30 minutes and then heat-shocked for exactly 30 seconds in a 42°C 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 37°C for 1 hour at 220rpm.