Difference between revisions of "Team:Aix-Marseille/Experiments/Protocols"
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===Preparation of the Bacterial culture=== | ===Preparation of the Bacterial culture=== | ||
# Recuperate the overnight bacterial culture | # Recuperate the overnight bacterial culture | ||
| − | # Determine the OD600 in a 1 | + | # Determine the OD600 in a 1 mL Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB |
# In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1 | # In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1 | ||
# Put the Erlenmeyer in an incubator for about an hour at 37˚C. | # Put the Erlenmeyer in an incubator for about an hour at 37˚C. | ||
| Line 59: | Line 59: | ||
|} | |} | ||
| − | Some solution aren’t available directly and are prepared | + | Some solution aren’t available directly and are prepared by dissolving the solid compound |
{| | {| | ||
| Line 147: | Line 147: | ||
#Hydrate by injecting 10µL of ddH2O | #Hydrate by injecting 10µL of ddH2O | ||
#Let it soak for 5 minutes until red coloration is highly visible | #Let it soak for 5 minutes until red coloration is highly visible | ||
| − | # | + | #pipet up-down to homogenize the solution |
| − | #Transfer the solution into an eppendorf set it on ice | + | #Transfer the solution into an eppendorf tube set it on ice |
No special conditions needed when manipulating just respect sterility | No special conditions needed when manipulating just respect sterility | ||
===Transformation of bacterial cells with plasmid=== | ===Transformation of bacterial cells with plasmid=== | ||
| − | #2 µL of | + | #2 µL of plasmid are transferred in eppendorf tube, excess are conserved at -20˚C |
#Add 100 µL of competent bacterial cells | #Add 100 µL of competent bacterial cells | ||
#Put on ice for 20 minutes | #Put on ice for 20 minutes | ||
| Line 162: | Line 162: | ||
#Centrifuge for 5 minutes at 5000rpm (normal centrifuge will work) | #Centrifuge for 5 minutes at 5000rpm (normal centrifuge will work) | ||
#Remove 400 µl of supernatant | #Remove 400 µl of supernatant | ||
| − | #Re-suspend | + | #Re-suspend pellet in the 150 µL of remaining medium pipet up-down |
#Spread total volume on petri dish with the appropriate antibiotic | #Spread total volume on petri dish with the appropriate antibiotic | ||
#Put petri dishes in an incubator overnight at 37˚C | #Put petri dishes in an incubator overnight at 37˚C | ||
| + | For a negative control repeat procedure without adding plasmid | ||
Handling is done near an open flame, sterile environment | Handling is done near an open flame, sterile environment | ||
Revision as of 21:26, 10 June 2017
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Protocols
Preparation of competent Bacterial cells
Preparation of the Bacterial culture
- Recuperate the overnight bacterial culture
- Determine the OD600 in a 1 mL Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB
- In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
- Put the Erlenmeyer in an incubator for about an hour at 37˚C.
- Test the OD600 for the new culture
- The OD600 should be between 0.4 and 0.6
All the handling done in a 15cm radius of an open flame for optimal sterility
Preparation of Buffer Tbf1 and Tbf2
| Tbf1 buffer | Total volume 80mL |
|---|---|
| KAc 1M | 2.4 mL |
| MnCl2 0.5M | 8 mL |
| KCl 1M | 8 mL |
| CaCl2 0.1M | 8 mL |
| GlY 80% | 15mL |
| H2O | 38.6 mL |
| Tbf2 buffer | Total volume 8 mL |
|---|---|
| NaMOPS 0.2 M | 400 µL |
| CaCl2 0.1 M | 6 mL |
| Gly 80% | 1.5 mL |
| KCl 1 M | 80 µL |
| H2O | 500 µL |
Some solution aren’t available directly and are prepared by dissolving the solid compound
| Volume (mL) | Mass (g) | Molecular weight (g/mol) | |
|---|---|---|---|
| MnCl2 0.5 M | 200 | 19.791 | 197.91 |
| KCl 1 M | 200 | 17.91 | 74.55 |
| NaMOPS 0.2 M | 50 | 2.0926 | 209.26 |
| KAc 1M | 50 | 4.9 | 98.15 |
Sterilize the flask prepared after weighing in an autoclave. When preparing Tbf1 and Tbf2 should be in proximity of an open flame
Preparation of Competent Bacterial cells
- Transfer the Bacterial culture in 50 ml Falcon tubes
- Centrifuge for 10 minutes at 3500 rpm in cold
- Remove the supernatant then re-suspend the pellet in 20 ml of Tbf1 for 50 ml of bacteria
- Centrifuge for 5 minutes at 3500 rpm in cold
- Poll in all bacterial culture in a single tube
- Remove the supernatant then re-suspend the pellet in 8 ml of Tbf2 for 200 ml of bacteria
- Allocate 220 µl of competent Bacterial cells in each eppendorf tube
- Instant freeze the eppendorf tubes in liquid nitrogen
- Conserve the tubes at -80˚C
NOTE: all handling done in a cold room. To re-suspend the pellet a hard jerking action applied on the tube. Alternatively, put on wheel for 10 minute.
Transformation protocol
Sensitivity test of competent bacterial cells
- Heat up LB-agar in micro oven at 300Watt for 19 minutes for 400 mL of LB-agar loosen cap before heating to let steam out
- Transfer 80 mL of LB-agar in to Erlenmeyer one for every antibiotic
- Pour 20 mL and spread the LB-agar in petri dishes, let it dry out
- Prepare petri dishes:
25µL of competent strain spread with balls in all four antibiotic and one without antibiotic as negative test
| Antibiotic stock | Desired concentration | Volume of antibiotic per mL of LB-agar | Volume in Erlenmeyer |
|---|---|---|---|
| Ampicillin 25 mg/mL |
100 µg/mL | 4 µL/mL | 320 µL |
| Kanamycin 10 mg/mL |
50 µg/mL | 5 µL/mL | 400 µL |
| Tetracycline 15 mg/mL |
15 µg/mL | 1 µL/mL | 80 µL |
| Chloramphenicol 30 mg/mL |
50 µg/mL | 1.6 µl/mL | 128 µL |
Handling is done near an open flame, sterile environment
Preparation of DNA material from iGEM kit
- Pierce plate at the desired location
- Hydrate by injecting 10µL of ddH2O
- Let it soak for 5 minutes until red coloration is highly visible
- pipet up-down to homogenize the solution
- Transfer the solution into an eppendorf tube set it on ice
No special conditions needed when manipulating just respect sterility
Transformation of bacterial cells with plasmid
- 2 µL of plasmid are transferred in eppendorf tube, excess are conserved at -20˚C
- Add 100 µL of competent bacterial cells
- Put on ice for 20 minutes
- Put tubes in thermomixer at 42˚C for 45 seconds
- Put on ice for 5 minutes
- Add 450 µL of LB
- Incubate tube for 1 hour at 37˚C
- Centrifuge for 5 minutes at 5000rpm (normal centrifuge will work)
- Remove 400 µl of supernatant
- Re-suspend pellet in the 150 µL of remaining medium pipet up-down
- Spread total volume on petri dish with the appropriate antibiotic
- Put petri dishes in an incubator overnight at 37˚C
For a negative control repeat procedure without adding plasmid Handling is done near an open flame, sterile environment
