Team:Paris/Notebook/Protocols
From 2008.igem.org
(→Protocol to make competent bacteria) |
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- | <br>1. Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a toothpick in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium. Over Night culture at 37°C | + | <br>1. Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a toothpick in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium. Over Night culture at 37°C / 300 rpm |
<br>2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL | <br>2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL | ||
- | <br>3. Culture at 37°C untill OD<sub>600</sub> reach 0.6 | + | <br>3. Culture at 37°C / 300 rpm untill OD<sub>600</sub> reach 0.6 |
<br>4. Fast cooling at +4°C by gently shaking the erlen in ice | <br>4. Fast cooling at +4°C by gently shaking the erlen in ice | ||
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<br>5. Use pre-cooled centrifuge at +4°C. Centrifuge 1mL of the culture in 1.5 mL tube: +4°C / 5 min / 5000 rpm | <br>5. Use pre-cooled centrifuge at +4°C. Centrifuge 1mL of the culture in 1.5 mL tube: +4°C / 5 min / 5000 rpm | ||
<br>6. Discard flowtrough by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down | <br>6. Discard flowtrough by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down | ||
- | <br>7. Add CaCl<sub>2</sub> QSP 20 mL and incubate 30 min / +4°C | + | <br>7. Add cold CaCl<sub>2</sub> QSP 20 mL and incubate 30 min / +4°C |
<br>8. Centrifuge 1mL of the suspension in 1.5 mL tube: +4°C / 5 min / 5000 rpm | <br>8. Centrifuge 1mL of the suspension in 1.5 mL tube: +4°C / 5 min / 5000 rpm | ||
<br>9. Discard flowtrough by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down | <br>9. Discard flowtrough by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl<sub>2</sub> and mix gently the suspension by up and down | ||
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<br>12. Incubate 20 min at +4°C | <br>12. Incubate 20 min at +4°C | ||
<br>13. Heat-Shock: 30-40 sec at 42°C / then 1 min in ice | <br>13. Heat-Shock: 30-40 sec at 42°C / then 1 min in ice | ||
- | <br>14. Add the sample to 1 mL LB medium without antibiotic and incubate 1h at 37°C | + | <br>14. Add the sample to 1 mL LB medium without antibiotic and incubate 1h at 37°C / 300 rpm |
<br>15. Spread 10 to 100 µL on plates with LB medium and the appropriate antibiotic | <br>15. Spread 10 to 100 µL on plates with LB medium and the appropriate antibiotic | ||
- | <br>16. Incubate O/N at 37°C | + | <br>16. Incubate O/N at 37°C / 300 rpm |
<br>17. Prepare a [https://2008.igem.org/Team:Paris/Notebook/Protocols#Glycerol_Stocks Glycerol Stock] or/and use the transformed bacteria to study [https://2008.igem.org/Team:Paris/Notebook/Protocols#Study_of_the_doubling_time_of_the_bacteria_population the doubling time of the bacteria population] | <br>17. Prepare a [https://2008.igem.org/Team:Paris/Notebook/Protocols#Glycerol_Stocks Glycerol Stock] or/and use the transformed bacteria to study [https://2008.igem.org/Team:Paris/Notebook/Protocols#Study_of_the_doubling_time_of_the_bacteria_population the doubling time of the bacteria population] | ||
Revision as of 16:16, 15 August 2008
Culture of Stable strain with biobricks 2008
Glycerol Stocks
Minipreps (Kit Qiagen)
ElectrophoresisAn electrophoresis can be done to check if there is Product of Miniprep
Concentration of the MiniprepBy biophotometry
Check if the ratio 260/280 is over 1,6 Digestion
Migration after digestion
| Separate each band by an empty one | !
Extraction
Amplification of promoters=>To amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
For each samples,
1 µl dNTP
LID : 105°C Purification (Kit Promega)Gel Slice and PCR Product PreparationDissolving the Gel Slice
Processing PCR reactionsFor products above 40 pb
Binding of DNA
Washing
Elution
Quantification by electrophoresis
Ligation
TransformationUse of TOP10 chemically competent cells
PCR ScreeningUse of 8 clones of Ligation transformants for screening PCR
After, add
Store the tubes on ice waiting for PCR attains 95°C then put the tubes in the machine
LID 105°C
Electrophoresis Purification of PCR
Sequencingvoir ici -> Sequencing COCHIN
Promoter Characterization PlanFor theoretical consideration, see estimation of parameters
The same colour coded steps can be perfomed at the same time if elements needed are available The order for treating the colours should of course be:
This table contains the promoters we need to characterize, the transcription factors whose effect on the promoter we want to test, and the plasmid we want to obtain in order to carry out each characterization
Protocol to make competent bacteria
Before: prepare CaCl2 0.1M.
Study of the doubling time of the bacteria population
- B1 Vitamin 0.1%
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