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Team:USTC/Standard & Protocol
From 2009.igem.org
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5. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) 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. | 5. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) 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. | ||
| + | ===Hybrid promoter response to AHL=== | ||
| + | 1. Streak a LB plate of the strain which contain one of the parts listed in pSB1A3 . | ||
| + | |||
| + | 2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic(Ampicillin 0.1mg/ml) with single colony from the plate. | ||
| + | |||
| + | 3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm. | ||
| + | |||
| + | 4. Cultures were diluted 1:1000 to tubes of 3ml fresh medium and grown for 4.5hrs. | ||
| + | |||
| + | 5. Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine is diluted and added to different tubes to yield different final concentrations (1E-5,1E-7,1E-8,1E-9,1E-10M).To ensure the same response time , the AHL should be added with a time interval of 2mins between tubes, so do the measurements procedure. | ||
| + | |||
| + | 6. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance ((HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell path length 10mm,600nm,1.5 nm slit width) for the first time 30 minutes after adding AHL. Repeat measurement every 30 mins in the next 4hrs. | ||
Revision as of 17:22, 20 October 2009
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Team:USTC/Standard & Protocol
Contents |
Standard protocol
Minipreps
Performed with BIO BASIC INC. EZ-10 Spin Column Plasmid DNA MiniPreps Kit BS414
Digestion
The digestion enzymes we use are listed:
Pst I Fermentas ER0611 3000U
EcoR I Fermentas ER0271 5000U
Spe I Fermentas ER1251 1500U
Xba I Fermentas ER0681 400U
Gel Extraction
Performed with BIO BASIC INC. EZ-10 Spin Column DNA Gel Extraction Kit BS354
Ligation
For short segments:
TakaRa DNA Ligation Kit Ver2.0 Code D6022
BIO BASIC INC. FAST LIGATION KIT BS512
For long segments ligation:
TaKaRa DNA Ligation Kit LONG Code D6024
Transformation
Colony PCR
Measurement protocol
Promoter measurements
1. Streak a LB plate of the strain which contain one of the parts listed in pSB1A3 .
2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic( Ampicillin 0.1mg/ml) with single colony from the plate.
3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） 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(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) 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.
Hybrid promoter response to AHL
1. Streak a LB plate of the strain which contain one of the parts listed in pSB1A3 .
2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic(Ampicillin 0.1mg/ml) with single colony from the plate.
3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm.
4. Cultures were diluted 1:1000 to tubes of 3ml fresh medium and grown for 4.5hrs.
5. Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine is diluted and added to different tubes to yield different final concentrations (1E-5,1E-7,1E-8,1E-9,1E-10M).To ensure the same response time , the AHL should be added with a time interval of 2mins between tubes, so do the measurements procedure.
6. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance ((HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell path length 10mm,600nm,1.5 nm slit width) for the first time 30 minutes after adding AHL. Repeat measurement every 30 mins in the next 4hrs.
