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<li>100 mL Bacterial Culture (DO=0,5-0,6)</li> | <li>100 mL Bacterial Culture (DO=0,5-0,6)</li> |
Revision as of 14:56, 22 October 2017
Protocols
Facing back to the Etendard glacier, in between the peaks of Maurienne and the ones of Oisan.
Credits: Estelle Vincent
Credits: Estelle Vincent
PREPARATIONS
LB Broth
Materials
Methods
- Add 20g of LB Broth Base per 1L of distillated water
- Homogenize
- Autoclave at 121°C for 15 minutes
CLOSE
LB Agar
Materials
Methods
- Add 32g of LB Agar per 1L of distilled water
- Homogenize
- Autoclave at 121°C for 15 minutes
CLOSE
Chloramphenicol (Cam) 25ug/mL Stock
Materials
Methods
- Add 0,25g of Cam per 10mL of ethanol
- Homogenize by vortexing
- Filter with a 22mm membrane filter
- Conserve at -20°C in 1mL aliquot
CLOSE
Petri Dish Preparation
Materials
Methods
- Add of 20uL Cam in 20mL of liquid LB Agar
- Pour the solution in the plate
- Wait until the agar solidifies
- Conserve returned in 4°C
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Glycerol 80%
Materials
Methods
- Harvest 80mL of glycerol 100%
- Add 20mL of sterile water
- Conserve at 4°C
CLOSE
CaCl2 100mM
Materials
Methods
- Dissolve 0,056g of CaCl2 in 5mL of sterile water
- Conserve at 4°C
CLOSE
MgCl2 100mM
Materials
Methods
- Dissolve 0,610g of MgCl2 in 30mL of sterile water
- Conserve at 4°C
CLOSE
Sucrose 100mM
Materials
Methods
- Dissolve 0,171g of sucrose in 5mL of sterile water
- Conserve at 4°C
CLOSE
IPTG 4mM
Materials
Methods
- Add 10µL of Cam in 10mL of LB
- Homogenize
- Remove 90µL of solution and add 80µL of IPTG
- Conserve at -20°C
CLOSE
Resuspension Solution for Lyophilized Bacteria
Materials
Materials
Methods
- Add 350µL of DMSO and 1,5µL of 2-mercaptoethanol in 5mL of sterile water
- Conserve at 4°C
CLOSE
EXPERIMENTATIONS
Competant bacteria
Standard protocol
Materials
Methods
- Centrifuge the culture at 5000rpm for 10 minutes at 4°C
- Remove the supernatant and resuspend pellet in 20mL of cold MgCl2
- Incubate 30 minutes on ice
- Centrifuge at 4000rpm for 10 minutes at 4°C
- Remove the supernatant and resuspend pellet in 2mL of CaCl2 and Glycérol 15%
- Aliquot 50uL in eppendorf previously cooled at -80°C
- Conserve at -80°C
Protocol for lyophilisation
Materials
Methods
- Centrifuge the culture at 4000rpm for 10 minutes at 4°C
- Remove the supernatant and resuspend pellet in 20mL of MgCl2
- Centrifuge at 4000rpm for 10 minutes at 4°C
- Remove the supernatant and resuspend pellet in 2mL of CaCl2 + Sucrose
- Aliquot 500µL by sterile ampoules
- Conserve at -80°C
CLOSE
Electrophorese
Materials
Methods for a 1% agarose gel
- Gel preparation
- Dissolve 1g of agarose in 100mL of TAE (1X)
- Pour the solution into the gel mould
- Let the solution gel (almost 15 minutes)
- Preparation of the samples to deposit
Add Loading Dye 6X to dilute it until 1X (usually 2µL added to 10µL sample)
NB: Maximum volume in the well is around 25µL & Minimum quantity of DNA detectable is around 25µg (for plasmid >3kb) - The migration is done at 100V during 30 minutes
- Revelation
- Prepare 250mL of distilled water in a tank
- Add 25uL of Gel Red. Do not forget to use gloves to manipulate the Gel Red
- Incubate the gel in the solution for 10 minutes to 1h protected from light
- Wash the gel in a tank of distilled water
- Read under UV
CLOSE
Bacterial Transformation
Materials
Methods
NB : Work under Microbiological Safety Bench and on ice
- Add DNA in 25µL of competent bacteria
- Gently invert the tube 4-5 times to mix cells and DNA. Do not vortex
- Incubate 30 minutes on ice
- Heat shock at 42°C for 1 minutes. Do not mix
- Place on ice for 2 minutes. Do not mix
- Incubate at 37°C and 150rpm for 2 hours
- Mix the cells thoroughly by inverting the tube
- Deposit 50µL on the first plate
- Centrifuge at 10000rpm for 3 minutes at Roof Temperature
- Remove a part of the supernatant and resuspend pellet with the rest
- Deposit the mixing on the second plate
- Incubate overnight at 37°C with plates upside down
CLOSE
Miniprep, Maxiprep
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PCR
Materials
Methods
- Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin
- Transfer reaction quickly to a preheated thermocycler at 98°C
- Thermocycling conditions
- Initial denaturation at 98°C during 30 seconds
- 35 Cycles : 10 seconds at 98°C - 30 seconds at 57°C - 30 seconds at 72°C
- Final extension at 72°C during 2 minutes
- Conserve at -20°C
CLOSE
Probe insertion
Materials
Methods
- EcoRI Plasmid Digestion
- Mix the plasmid DNA, 1,3µL of EcoRI, 5µL of buffer and 40,7µL of distilled water
- Incubate 15 minutes at 37°C
- Dephosphorylation
- Add 1µL of CIAP
- Incubate 5 minutes at 37°C
- Incubate 15 minutes at 65°C
NB : This steep can be repeated until the plasmid is not ligature on itself - Control by ligation and bacterial transformation
- Prepare 1µL of the digested and dephosphorylated plasmid
- Add 1µL of ligase and 2µL of its buffer and 16µL of distilled water
- Incubate 10 minutes at Roof Temperature
- Incubate 10 minutes at 65°C for inactivation
- Transformation (cf. protocols)
NB : This steep is repeated in order to have the minimum of colonies - EcoRI Probe Digestion
- Mix 1µL of probe DNA, 0,5µL of EcoRI, 2µL of CutSmart Buffer and 16,5µL of distilled water
- Incubate 15 minutes at 37°C
- Incubate 20 minutes at 65°C for inactivation
- Ligature
- Preheat tube at 70°C to limit the hydrogen bonded
- Add 0,3µL of the preparation containing the probe, 2µL of the dephosphorylated plasmid (100ng), 1µL of ligase, 2µL of its buffer and 15µL of distilled water
- Incubate 10 minutes at Roof Temperature
- Incubate 10 minutes at 65°C for inactivation
CLOSE
Plasmid activation
Materials
Methods
- Prepare 2µg of backbone DNA in a tube
- Add 4µL of BmtI with 2µL of NEBuffer 3.1. and 12,4µL of water
- Incubate 1 hour at 37°C
- Incubate 20 minutes at 65°C for inactivation
- Add 2µL of BglI with 1µL of NEBuffer 3.1. and 7µL of water
- Incubate 15 minutes at 37°C
- Add 2µL of Nt.BspQI with 1µL of NEBuffer 3.1. and 7µL of water
- Incubate 1 hour at 50°C
- Incubate 20 minutes at 80°C for inactivation
- Add 2µL of Nt.BtsI with 5µL of CutSmart Buffer and 3µL of water
- Incubate 1 hour at 37°C
- Incubate 20 minutes at 80°C for inactivation
CLOSE
Purification and DNA extraction from agarose gel
Were carried out according to the NucleoSpin Gel® and PCR Clean-up Kits
CLOSE
Plasmid drying
Materials
Methods
- Put 15µL of plamid in the Speed Vac
- Let it for 45 minutes
- Resuspended in 15uL of distillated water
CLOSE
Bacteria lyophilisation and resuspension
Materials
Methods
NB : Work under Microbiological Safety Bench and on ice
- Lyophilize the bacteria coming from the protocol “Competent Bacteria for Lyophilisation”
- Conserve at 4°C
- Resuspension
- Resuspend the lyophilised bacteria with 500uL of the resuspension solution
- Incubate 10 minutes on ice
- Use or conserve the bacteria at -20°C
CLOSE