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

0,056g 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 0,610g 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

Electrophorese

Materials

1g Agarose (powder)

100mL TAE

Distilled Water

25µL Gel Red

Methods for a 1% agarose gel

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
   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)
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 to 1h protected from light
4. Wash the gel in a tank of distilled water
5. Read under UV

Bacterial Transformation

Materials

25uL DH5a Competent Cells

1 to 5µL DNA (concentration>20ng/µL)

450µL SOC

2 Petri dish

Methods
NB : Work under Microbiological Safety Bench and on ice

1. Add DNA in 25µL of competent bacteria
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 (NEB)

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

PCR

Materials

2,5µL Primers Forward (10µM)

2,5µL Primers Reverse (10µM)

25µL Polymerase Mix

10ng Probe DNA (1µL)

Sterile Water sat (sufficient amount to) 50µL

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

Preparation of the plasmid backbone

Materials

2,5µg Plasmid DNA

50U EcoRI (2,5µL@20 000U/mL)

5µL CutSmart buffer 10X

50µL Distilled Water

3U CIAP (Calf Intestinal Alkaline Phosphatase, 3µL@ 1U/µL)

1200 U T4 DNA Ligase (3µL@400 000/mL)e

6µL T4 DNA Ligase Reaction Buffer 10X

Methods

1. Plasmid Digestion
1. Mix the plasmid DNA, EcoRI, the CutSmart buffer and distilled water until 50µL final volume
2. Incubate 15 minutes at 37°C
2. Serial dephosphorylations
1. In each sample to dephosphorylate: add 1U (1µL) of CIAP
2. Incubate 5 minutes at 37°C
3. Incubate 15 minutes at 65°C (for inactivation of the phosphatase)
   Step is repeated three times (to ensure the avoidance of self-ligation).
   NB: At each step, sample is divided in two: the part that goes through a new dephosphorylation and the one that does not.
3. Control by ligation (without the probe) and bacterial transformation
1. Prepare 1uL (50ng) of the digested and X-times dephosphorylated plasmid
2. Add 1uL of ligase, 2uL of its buffer and distilled water sat 20µL
3. Incubate 10 minutes at Room Temperature
4. Incubate 10 minutes at 65°C for inactivation
5. Transformation (cf. upstream protocol)

Preparation of the probe

Materials

300ng Probe DNA (1µL@315ng/µL)

10U EcoR1 (0,5µL @20 000U/mL)

2µL CutSmart Buffer 10X

5Distilled water sat 20µL

Methods : Probe digestion

1. Mix the probe DNA, EcoRI, the CutSmart Buffer and the distilled water until 20µL final volume
2. Incubate 15 minutes at 37°C
3. Incubate 20 minutes at 65°C for inactivation

Ligation Probe - Plasmid backbone

Materials

12µL of the plasmid DNA digested and dephosphorylated 3 times

4,7µL of the probe digested

5µL T4 DNA Ligase (@400 000/mL)

12µL T4 DNA Ligase 10X

120µL distilled water

Methods

1. Insertion probe
1. Preheat tubes probe and plasmid backbone at 70°C to limit the hydrogen bounds
2. Prepare the following mix (help: NEBBioCalculator)

Molar ratio probe/plasmid	0:1	1:1	3:1	7:1	10:1	3:1 negative control without ligase
Plasmid (dephosphorylated 3 times @50ng/µL)	2µL
Probe (@15,7ng/µL)	0	0,3µL	0,6µL	1,3µL	1,9µL	0,6µL
T4 DNA ligase (@400 000/mL)	1µL					0
Buffer T4 Ligase 10X	2µL
Distilled Water	Complete to 20µL final volume

3. Incubate 10 minutes at Room Temperature
4. Incubate 10 minutes at 65°C for inactivation
2. Transformation
Transform 1µL of each product of ligation in 20µL competent DH5α.
See protocol upstream.

Screening

Materials

75mL LB+cam media

15 miniprep columns (Monarch® Plasmid Miniprep Kit (NEB)

15µL EcoR1 (@20 000U/mL)

30µL Cutsmart buffer 10X

300 µL distilled water

2g agarose

600mL TAE 1X

Methods

1. Pick up 3 colonies per conditions of ligation (ratio 1:1 to 7:1 + neg control w/o ligase) to put them separately in 5mL LB+cam
2. Amplify via overnight culture (37°C, agitation)
3. Extract DNA from each cultures
4. Digest 1µg of each DNA extract with EcoR1 (20U enzyme, 2µL CutSmart Buffer 10X, water until 20µL final volume and incubation 15’ at 37°C)
5. Prepare a 2% agarose gel (2g agarose for 100mL TAE 1X) (see protocol above)
6. Migrate the digested DNAs (see protocol above)
7. Check which plasmid backbone inserted the probe (should appear at 96bp)

CLOSE

Purification and DNA extraction from agarose gel

Were carried out according to the NucleoSpin Gel® (MN) and PCR Clean-up Kits (Invitrogen)

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 for Lyophilization

500µL Resuspension Solution for Lyophilized Bacteria

Methods
NB : Work under Microbiological Safety Bench and on ice

1. Lyophilize the bacteria
2. Conserve at 4°C
3. Resuspend the lyophilised bacteria with 500uL of the resuspension solution
4. Use or conserve the bacteria at -20°C