Difference between revisions of "Team:Grenoble-Alpes/Protocols"

Preparations

LB Broth

Materials

20g LB Broth Base (powder)

1L Distillated Water

Methods

1. Add 20g of LB Broth Base per 1L of distillated water
2. Homogenize
3. Autoclave at 121°C for 15 minutes

LB Agar

Materials

32g LB Agar (powder)

1L Distilled Water

Methods

1. Add 32g of LB Agar per 1L of distilled water
2. Homogenize
3. Autoclave at 121°C for 15 minutes

Chloramphenicol (Cam) 25ug/mL Stock

Materials

0,25g Chloramphenicol (25mg/mL)

10mL Ethanol

Methods

1. Add 0,25g of Cam per 10mL of ethanol
2. Homogenize by vortexing
3. Filter with a 22mm membrane filter
4. Conserve at -20°C in 1mL aliquot

Petri Dish Preparation

Materials

20mL LB Agar

20µL Cam (25µg/mL)

Methods

1. Add of 20uL Cam in 20mL of liquid LB Agar
2. Pour the solution in the plate
3. Wait until the agar solidifies
4. Conserve returned in 4°C

Glycerol 80%

Materials

80mL Glycerol 100%

20mL Sterile Water

Methods

1. Harvest 80mL of glycerol 100%
2. Add 20mL of sterile water
3. Conserve at 4°C

CaCl2 100mM

Materials

0,0555g CaCl2 (111g/mol)

5mL Sterile Water

Methods

1. Dissolve 0,056g of CaCl2 in 5mL of sterile water
2. Conserve at 4°C

MgCl2 100mM

Materials

0,610g MgCl2 (203,31g/mol)

30mL Sterile Water

Methods

1. Dissolve 0,6105g of MgCl2 in 30mL of sterile water
2. Conserve at 4°C

Sucrose 100mM

Materials

0,171g Sucrose

5mL Sterile Water

Methods

1. Dissolve 0,171g of sucrose in 5mL of sterile water
2. Conserve at 4°C

IPTG 4mM

Materials

80µL IPTG (500mM)

10mL LB

10µL Cam (25ug/mL)

Methods

1. Add 10µL of Cam in 10mL of LB
2. Homogenize
3. Remove 90µL of solution and add 80µL of IPTG
4. Conserve at -20°C

Resuspension Solution for Lyophilized Bacteria

Materials

350µL DMSO

1,5µL 2-mercaptoethanol 1%

5mL Sterile Water

Methods

1. Add 350µL of DMSO and 1,5µL of 2-mercaptoethanol in 5mL of sterile water
2. Conserve at 4°C

Experimentations

Competant bacteria for lyophilisation

Materials

100 mL Bacterial Culture (DO=0,5-0,6)

20mL MgCl2 (100mM)

2mL CaCl2 (100mM) + Sucrose (100mM)

Sterile Ampoules

Parafilm

Liquid Nitrogen

Methods

1. Centrifuge the culture at 4000rpm for 10 minutes at 4°C
2. Remove the supernatant and resuspend pellet in 20mL of MgCl2
3. Centrifuge at 4000rpm for 10 minutes at 4°C
4. Remove the supernatant and resuspend pellet in 2mL of CaCl2+Sucrose
5. Aliquot 500µL by sterile ampoules
6. Cover with drilled parafilm
7. Freeze in liquid nitrogen
8. Conserve at -80°C

Electrophorese

Materials

1g Agarose (powder)

100mL TAE

Distilled Water

25µL Gel Red

Methods

1. Gel preparation
1. Dissolve 1g of agarose in 100mL of TAE (1X)
2. Pour the solution into the gel mould
3. Let the solution gel (almost 15 minutes)
2. Preparation of the samples to deposit
3. The migration is done at 100V during 30 minutes
4. Revelation
1. Prepare 250mL of distilled water in a tank
2. Add 25uL of Gel Red. Do not forget to use gloves to manipulate the Gel Red
3. Incubate the gel in the solution for 10 minutes at the obscurity
4. Wash the gel in a tank of distilled water

Bacterial Transformation

Materials

25uL DH5a Competent Cells

1,5µL DNA

450µL SOC

2 Petri dish

Methods
NB : Work under Microbiological Safety Bench and on ice

1. Add 1,5µL of DNA in 25µL
2. Gently invert the tube 4-5 times to mix cells and DNA. Do not vortex
3. Incubate 30 minutes on ice
4. Heat shock at 42°C for 1 minutes. Do not mix
5. Place on ice for 2 minutes. Do not mix
6. Incubate at 37°C and 150rpm for 2 hours
7. Mix the cells thoroughly by inverting the tube
1. Deposit 50µL on the first plate
2. Centrifuge at 10000rpm for 3 minutes at Roof Temperature
3. Remove a part of the supernatant and resuspend pellet with the rest
4. Deposit the mixing on the second plate
8. Incubate overnight at 37°C with plates upside down

Miniprep, Maxiprep

Minipreps were carried out according to the Monarch® Plasmid Miniprep Kit

Maxipreps were carried out according to the Pure Link® HiPure Plasmid DNA Purification Kits

PCR

Materials

2,5µL Primers Forward (10µM)

2,5µL Primers Reverse (10µM)

25µL Polymerase Mix

1µL Probe DNA

19µL Sterile Water

Methods

1. Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin
2. Transfer reaction quickly to a preheated thermocycler at 98°C
3. Thermocycling conditions
1. Initial denaturation at 98°C during 30 seconds
2. 35 Cycles : 10 seconds at 98°C - 30 seconds at 57°C - 30 seconds at 72°C
3. Final extension at 72°C during 2 minutes
4. Conserve at -20°C

Probe insertion

Materials

2,5µL Plasmid DNA

1,8µL EcoRI

7µL EcoRI Buffer

88µL Distilled Water

1µL CIAP

2µL T4 DNA Ligase

4µL T4 DNA Ligase Reaction Buffer (10X)

1µL Probe DNA

Methods

1. EcoRI Plasmid Digestion
1. Mix the plasmid DNA, 1,3µL of EcoRI, 5µL of buffer and 40,7µL of distilled water
2. Incubate 15 minutes at 37°C
2. Dephosphorylation
1. Add 1µL of CIAP
2. Incubate 5 minutes at 37°C
3. Incubate 15 minutes at 65°C
   NB : This steep can be repeated until the plasmid is not ligature on itself
4. Control by ligation and bacterial transformation
1. Prepare 1µL of the digested and dephosphorylated plasmid
2. Add 1µL of ligase and 2µL of its buffer and 16µL of distilled water
3. Incubate 10 minutes at Roof Temperature
4. Incubate 10 minutes at 65°C for inactivation
5. Transformation (cf. protocols)
   NB : This steep is repeated in order to have the minimum of colonies
3. EcoRI Probe Digestion
1. Mix 1µL of probe DNA, 0,5µL of EcoRI, 2µL of CutSmart Buffer and 16,5µL of distilled water
2. Incubate 15 minutes at 37°C
3. Incubate 20 minutes at 65°C for inactivation
4. Ligature
1. Preheat tube at 70°C to limit the hydrogen bonded
2. 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
3. Incubate 10 minutes at Roof Temperature
4. Incubate 10 minutes at 65°C for inactivation

Plasmid activation

Materials

2µg Backbone DNA (plasmid+probe)

4µL BmtI

2µL BglI

2µL Nt.BspQI

2µL Nb.BtsI

4µL NEBuffer 3.1. 10X

5µL CutSmart Buffer 10X

29,4µL Water

Methods

1. Prepare 2µg of backbone DNA in a tube
2. Add 4µL of BmtI with 2µL of NEBuffer 3.1. and 12,4µL of water
3. Incubate 1 hour at 37°C
4. Incubate 20 minutes at 65°C for inactivation
5. Add 2µL of BglI with 1µL of NEBuffer 3.1. and 7µL of water
6. Incubate 15 minutes at 37°C
7. Add 2µL of Nt.BspQI with 1µL of NEBuffer 3.1. and 7µL of water
8. Incubate 1 hour at 50°C
9. Incubate 20 minutes at 80°C for inactivation
10. Add 2µL of Nt.BtsI with 5µL of CutSmart Buffer and 3µL of water
11. Incubate 1 hour at 37°C
12. Incubate 20 minutes at 80°C for inactivation

Purification and DNA extraction from agarose gel

Were carried out according to the NucleoSpin Gel® and PCR Clean-up Kits

Plasmid drying

Materials

15µL Plasmid

15µL Sterile Water

Speed Vac

Methods

1. Put 15µL of plamid in the Speed Vac
2. Let it for 45 minutes
3. Resuspended in 15uL of distillated water

Bacteria lyophilisation and resuspension

Materials

500µL Competent Bacteria

500µL Resuspension Solution for Lyophilized Bacteria

Methods
NB : Work under Microbiological Safety Bench and on ice

1. Lyophilize the bacteria coming from the protocol “Competent Bacteria for Lyophilisation”
2. Conserve at 4°C
3. Resuspension
1. Resuspend the lyophilised bacteria with 500uL of the resuspension solution
2. Incubate 10 minutes on ice
4. Use or conserve the bacteria at -20°C