Team:USTC CHINA/Notebook/Protocols/Constitutive promoter measurements

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<h1>Gel Extraction</h1>
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<h1>Constitutive promoter measurements</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>1. Streak a LB plate of the strain.</br>
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Protocol
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2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic with single colony from the plate.</br>
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1. Excise gel slice containing DNA fragment of interest.
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3. Cultures were grown in test tubes for 16hrs at 37℃ with shaking at 200rpm.</br>
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2. Add 3×sample volume of Buffer DE-A.
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4. Cultures were diluted 1:100 into 3ml fresh medium and grown for 3hrs.</br>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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5. Measure the fluorescence (performed with SpectraMax M5 Multi-Mode Microplate Reader，add 200ul liquid in each well) and absorbance (HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell,path length 10mm,600nm,1.5 nm slit width) every 30 minutes in the next 4hrs.</br>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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Repeat wash with Buffer W2
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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Revision as of 16:00, 25 September 2013

Constitutive promoter measurements

1. Streak a LB plate of the strain.
2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic with single colony from the plate.
3. Cultures were grown in test tubes for 16hrs at 37℃ with shaking at 200rpm.
4. Cultures were diluted 1:100 into 3ml fresh medium and grown for 3hrs.
5. Measure the fluorescence (performed with SpectraMax M5 Multi-Mode Microplate Reader，add 200ul liquid in each well) and absorbance (HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell,path length 10mm,600nm,1.5 nm slit width) every 30 minutes in the next 4hrs.

notebook

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