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

CHLORAMPHENICOL (25µg/mL) stock

Materials

• 0.25g of Chloramphenicol
• 10mL of ethanol

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

AMPICILLIN (100 mg/mL) stock

Materials

• 1g of Ampicillin
• 10mL of ethanol

Methods

• Add 1g of Amp per 10mL of ethanol
• Homogenize by vortexing
• Filter with a 22mm membrane filter
• Store at -20°C in 1mL aliquot

Materials

• 100mL bacteria culture (OD = 0.5-0.6)
• 20mL MgCl2 (100mM)
• 2mL CaCl2 (100mM)
• Glycerol 15%

Methods

• Centrifuge the culture at 5000 rpm for 10 minutes at 4°C
• Remove the supernatant and resuspend the pellet in 20mL of cold MgCl2 100mM
• Incubate 30 minutes on ice
• Centrifuge at 4000 rpm for 10 minutes at 4°C
• Remove the supernatant and resuspend the pellet in 2mL of CaCl2 (100mM), Glycerol 15%
• Aliquot 50µL in Eppendorf previously cooled at -80°C
• Store at -80°C

Materials

• 25µl of DH5alpha competent cells
• 1 to 5µl of DNA (concentration > 20ng/µl)
• 450µl of SOC medium
• 2 Petri dishes

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 minute. Do not mix
• Place on ice for 2 minutes. Do not mix
• Add 450µL of SOC medium.
• Incubate at 37°C and 200rpm for 2h
• Mix the cells thoroughly by inverting the tube
• Deposit 50µl on the first plate
• Centrifuge at 1000rpm for 3 minutes at Room 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

Bacteriophage amplification via liquid medium

Material

• Liquid bacteriophage sample and its bacterial host grown in agar plate
• Luria-Bertani (LB) broth medium
• CaCl2 1M and MgCl2 1M solutions

Methods

• Grow bacterial host in LB broth (e.g. 5mL) overnight at 37 °C with agitation (200rpm).
• Prepare a bacterial culture with new LB broth adding 1/10 of overnight bacterial host (for example add 3,5mL of overnight
• bacterial host in 35mL of new LB broth medium). Mix.
• Incubate at 37°C with agitation (200rpm) a few hours or overnight.*
• Add a final concentration of 2mM of CaCl2 and MgCl2 (for example add 70µL of CaCl2 1M and 70µL of MgCl2 1M in the 35mL culture). Mix.
• Add 100µL of high titer bacteriophage stock (>108 pfu/mL) (the volume can be adapted according the titer).
• Mix.
• Incubate at 37°C with agitation until lysate clears (disappearance of the turbid) - approximately 5 hours -.
• Store at 4°C.
• The bacteriophage lysate titer can be measured to ensure the success of the amplification.

*It depends on the growth rate of the bacterial host

Bacteriophage purification

Material

• Bacteriophage lysate sample
• SM (Sodium chloride - Magnesium sulphate) Buffer
• PEG(20%)/NaCl(2.5M) solution
• NaCl 5M solution

Methods

• Add, to the bacteriophage lysate, a final concentration of 1M of NaCl (for example add 7mL of NaCl 5M solution in 35mL bacteriophage lysate). Mix.
• Centrifuge for 15 min at ⩾4,000 x rpm.
• Recover the supernatant.
• Add a minimum of 0.25 volume of PEG(20%)/NaCl(2.5M) solution (PEG 6000 or 8000 can be used) to the supernatant (e.g. add a minimum of 10mL of PEG/NaCl to for 40mL of supernatant) . Mix (do no vortex).
• Let mixed to the sample at room temperature for 20 min and overnight at 4°C. *
• Centrifuge at ⩾4,000 x rpm for 20 min at 4°C.
• Discard the supernatant.
• Resuspend the pellet in SM Buffer with 0.01-0.02 initial culture volume (e.g. add around 500µL for an initial bacterial culture of 50mL).**
• Let resuspend the pellet at 4°C for 1h minimum.*
• Store at 4°C

*The bacteriophage lysate titer can be measured (by Plaque Assay) to ensure the success of this step.
**For better purification, add 10mL of SM Buffer, do the next step, and repeat the steps 4 to 9.

Plaque assay for determination of bacteriophage titer

Material

• Bacteriophage sample requiring titering and its bacterial host grown in agar plate
• LB broth medium
• LB top agar
• SM Buffer
• Agar plate

Methods

• Grow bacterial host in LB broth (e.g. 5mL) at 37 °C with agitation (200 rpm) for few hours.
• Heat LB top agar in microwave until completely melted
• Allow top agar to cool until it is possible to touch the container (between 42 and 56°C).
• Do a serial dilution by diluting 10µL of bacteriophages stock in SM Buffer (90µL for 10-1 dilution or 990µL for 10-2 dilution) and repeat this step with the sample obtained until get the desired dilution.

Spot titration

• Spot titration is used to get a rough idea of the bacteriophage titer.
• Add 250µL of bacterial host for 5mL of LB top agar and mix.*
• Dispense the LB top agar-bacteria mixture onto an agar plate.
• Let gel.
• Spot 10µL of a bacteriophage dilution in a part of the plate and do the same for other dilutions. Until 10 dilutions can be spotted in one plate.
• Allow the liquid from the spots to absorb into the LB top agar (15 min).
• Invert the plate and incubate at 37°C overnight.

Full titration

• Full titration is used to get a accurate idea of the bacteriophage titer. It’s advisable to do a plate with the valid dilution (obtained with the spot titration) and the two nearest dilutions.
• Add 250µL of overnight bacterial host and 250µL of bacteriophage dilution for 5mL of LB top agar.*
• Mix and dispense the mixture onto an agar plate.
• Let gel and invert the plate.
• Incubate at 37°C overnight.

*Suggested quantities are valid for one plate.

SM buffer preparation (500mL)

Material

• 2.9g of NaCl
• 1g of MgSO₄⋅7H2O
• 25mL of Tris⋅HCl 1M pH7.4
• Distilled water to 500mL

Methods

• Add 2.9g of NaCl, 1g of MgSO4⋅7H₂O and 25mL of Tris⋅HCl 1M pH7.4 in 500mL of distilled water.
• Shake until dissolved.
• Autoclave.

NaCl 5M preparation (200mL)

Material

• 58.44 g of NaCl
• Distilled water to 200mL

Methods

• Add 58.44g of NaCl in 200mL of distilled water.
• Shake until dissolved.
• Autoclave.

PEG(20%)/NaCl(2.5M) preparation (200mL)

Materials

• 29.22g of NaCl
• 40g of PEG 6000 or PEG 8000
• Distilled water to 200mL

Methods

• Add 29.22g of NaCl, 40g of PEG 8000 (or PEG 6000) in 200mL of distilled water.
• Allow the PEG to completely dissolve, mixing frequently.
• Autoclave.

CaCl2 1M preparation (100mL)

Materials

• 14.70g of CaCl₂⋅2H₂O
• Distilled water to 100mL

Methods

• Add 14.70g of CaCl₂⋅2H₂O in 100mL of distilled water.
• Shake until dissolved.
• Autoclave.

MgCl2 1M preparation (100mL)

Materials

• 20.33g of CaCl₂⋅6H₂O
• Distilled water to 100mL

Methods

• Add 20.33g of CaCl₂⋅6H₂O in 100mL of distilled water.
• Shake until dissolved.
• Autoclave.

LB top agar preparation (500mL)

Materials

• 4g of agar-agar
• 1mL of CaClsub>2 1M
• 1mL of MgCl₂ 1M
• LB broth medium to 500mL

Methods

• Add 4g of agar-agar, 1mL of CaCl₂ 1M, 1mL of MgCl₂ 1M in 500mL of LB broth.
• Shake.
• Autoclave.

Tris⋅HCl 1M pH 7.4 preparation (100mL)

Materials

• 12.11g of Tris
• Concentrated HCl solution
• Distilled water to 100mL

Methods

• Add 12.11g of Tris in 30mL of distilled water.
• Shake until dissolved.
• Adjust slowly pH to 7.4 with the appropriate volume of concentrated HCl (helping with a pH meter).
• Add distilled water to 100mL
• Shake.
• Autoclave.

REFERENCES

Bonilla, N., Rojas, M. I., Netto Flores Cruz, G., Hung, S.-H., Rohwer, F., & Barr, J. J. (2016). Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. PeerJ, 4, e2261. doi:10.7717/peerj.2261

M. Poxleitner, W. Pope, D. Jacobs-Sera, V. Sivanathan & G. Hatful, 2017, Phage Discovery Guide.

Materials

• Lysis buffer
• Extraction buffer
• Elution buffer
• Magnetic beads
• Magnet
• Luria-Bertani (LB) broth medium
• CaCl₂ 1M and MgCl₂ 1M solutions
• Liquid bacteriophage sample (>1025 pfu/mL) and its bacterial host grown in agar plate.

Methods

• Grow bacterial host in LB broth (e.g. 5mL) overnight at 37 °C with agitation (200rpm).
• Add a final concentration of 2mM of CaCl₂ and MgCl2 (for example add 10µL of CaCl₂ 1M and 10µL of MgCl₂ 1M in the 5mL culture) and mix.
• The next steps are realized in eppendorfs with a volume of 200µL of bacterial host.
• Add 50µL of very high titer bacteriophage stock (>1025 pfu/mL) and mix.
• Incubate at 37°C with agitation approximately 4.5 hours.
• Add 20µL of magnetic beads and flush.
• Incubate 10 minutes at room temperature.
• Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
• Wash by adding 500µL of Extraction Buffer and flush.
• Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
• Wash again by adding 500µL of Extraction Buffer. Flush
• Remove the supernatant by keeping magnetic beads in the eppendorf using the magnet.
• Wash one last time by adding 500µL of Elution Buffer. Flush
• Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
• Add 30µL of Elution Buffer and flush.
• Incubate 5 minutes at 70°C.
• Remove the magnetic beads by sliding them along the wall of the eppendorf using the magnet.
• Retrieve the eluent containing the DNA using a pipette and deposit it in a new eppendorf.
• The DNA can be stored at -20°C.

Positive control:

• Add 400µL of Lysis Buffer in 200µL of bacterial host.
• Incubate 10min at room temperature.
• Add 20µL of magnetic beads
• Incubate 10min at room temperature
• Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
• Wash by adding 500µL of Lysis Buffer and flush.
• Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
• Continu by repeating from step 8. of the DNA extraction of bacteria lysed by bacteriophages.

Negative control (1):

Do the same protocol of extraction from bacteria lysed by phages but add 50µL of SM Buffer instead of 50µL of bacteriophages (step 3).

Negative control (2):

Replace the 200µL of bacterial host (step 1 of original protocol) by 200µL of LB broth medium and do the same protocol for the next.

Materials

• 1g of agarose (powder)
• 100mL TAE 1X
• Distilled water
• 25µL of GelRed

Methods (for a 1% agarose gel)

Gel preparation

• Dissolve 1g of agarose in 100mL of TAE (1X)
• Pour the solution into the gel mold
• 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 and minimum quantity of DNA detectable is around 25µg (for plasmid > 3Kb)

• The migration is done at 100V for 30 minutes

Revelation

• Prepare 250mL of distilled water in a tank
• Add 25µl 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

Materials

Materials (out of the NEB kit*):

• High-Fidelity DNA polymerase (Q5)
• Lysogeny broth (LB)
• SOC medium
• DH5α bacteria

Methods

Gibson assembly reaction

NB: All the Gibson experiments have to be done into ice.





*X μl: Optimized cloning efficiency requires about 50 – 100 ng of vector and at least 2-fold excess inserts. Use 5X more insert if the size is less than 200 bps.
• Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled. Following incubation, store samples on ice or at – 20°C for subsequent transformation.
• Transform competent cells with 2μl of the assembly reaction, using the following the transformation protocol.

Chemically Competent Cells Transformation Protocol

• Thaw chemically competent cells ices.
• Transfer 50 μl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
• If the chemically competent cells are from New England Biolabs, add 2 μl of assembled product to NEB competent cells.
• Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30min. Do not mix.
• Heat shock at 42°C for 45 seconds. Do not mix.
• Transfer tubes on ice for 2 minutes.
• Add 950 μl of room temperature SOC media to tubes.
• Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
• Warm selection plates to 37°C.
• Spread 100 μl of the cell onto the plates with appropriate antibiotics. Use Amp plates for a positive control sample.
• Incubate plates overnight at 37°C.

REFERENCES OF THE NEB KIT*

[1] http://www.thermofisher.com/order/catalog/product/K210004

[2] https://international.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information

[3] http://www.thermofisher.com/order/catalog/product/K210002

IPTG (4.5 mM)

Materials

• 0.95mg of IPTG
• 80µL of sterilized water
• 10mL LB
• 10uL Cam (25 µg/mL)

Methods

• Add 10µl of Cam in 10mL of LB
• Homogenize
• Add 0.95mg of IPTG in 80µl of sterilized water
• Homogenize
• Remove 90µl of Cam/LB mix and add 80µl of IPTG
• Conserve at -20°C

For a serial dilution

Materials

• 888.8µl of IPTG (4,5mM)

Methods

LB AGAR

Materials

• 32g of LB agar (powder)
• 1L of distilled water

Methods

• Add 32g of LB agar per 1L of distilled water
• Homogenize
• Autoclave at 121°C for 15 minutes

LB BROTH

Materials

• 20g LB Broth Base (powder)
• 1L distilled water

Methods

• Add 20g of LB Broth Base per 1L of distilled water
• Homogenize
• Autoclave at 121°C for 15 minutes

MINIPREP & MIDIPREP

Minipreps were carried out according to the NucleoSpin Plasmid Miniprep Kit (NEB).
Midipreps were carried out according to the NucleoBond XtraMidi (NEB).

PETRI DISH PREPARATION

Materials

• 20mL of LB agar
• 20µl of Cam (25 µg/mL)

Methods

• Add of 20µl Cam in 20mL of liquid LB agar
• Pour the solution in the plate
• Wait until the agar solidifies
• Conserve returned in 4°C

COMPETENT BACTERIA AND FREEZE-DRYING

Materials

• Commercial bacteria (DH5ɑ, TOP10 or BL21 E. Coli for example)
• 200mL of LB broth medium
• 20mL MgCl₂ 100mM
• 1mL CaCl₂ 200mM
• 1mL Carbohydrates solution (sucrose, trehalose) at desired concentrations (for example 200mM Sucrose)

Methods

• Plate some commercial bacteria on LB-agar without antibiotic and let grow overnight
• Start a preculture from one single colony in 8mL LB broth medium without antibiotic and incubate overnight at 37°C under agitation
• Start a large culture of 100mL-150mL LB broth with the preculture (depending on the number of bacteria you need) without adding antibiotic, incubate for at least 2h at 37°C under agitation and control O.D. 600nm until it reaches 0.7-0.8.
• Separate the culture volume in 50mL falcons and incubate in ice for at least 30minutes

NB:FROM THAT POINT, BACTERIA NEED TO STAY IN ICE AS MUCH AS POSSIBLE TO COMPLETE THE PROTOCOL EFFICIENTLY

• Centrifuge the culture at 5000 rpm for 10 minutes at 4°C
• Remove the supernatant and resuspend the pellet in 20mL of ice-cold MgCl₂ 100mM
• Incubate 30 minutes on ice
• Centrifuge at 4000 rpm for 10 minutes at 4°C
• Remove the supernatant and resuspend the pellet in 1mL of CaCl₂ 200mM
• Make aliquots of 25μL resuspended bacteria and complete with 25μL carbohydrate solution diluted in ice-cold sterile water to the desired concentration
• Snap-freeze them in liquid nitrogen before lyophilization protocol
• Conserve freeze-dried samples in a 4°C fridge

FREEZE-DRIED COMPETENT BACTERIA REHYDRATION

Materials

• Aliquots of competent bacteria freeze-dried as described in “Competent bacteria for freeze-drying” protocol (lien vers celui d’avant)
• Resuspension solution (sterile water, DMSO 7%) : 50μL per aliquot

Methods

• Put freeze-dried aliquots in ice and resuspend it with an ice-cold resuspension solution
• Flush for few minutes and make sure to resuspend all dried bacteria
• Incubate in ice for 10 minutes
• Flush again and you can then use bacteria as if they were conserved frozen

PLASMIDS DRYING

Materials

• Plasmid DNA extracted with a Macherey Nagel’s Midiprep kit in 300μL of sterile water, concentrated up to ~300ng/μL
• Sterile water

Methods

• Make 15μL plasmid DNA aliquots diluted to the desired concentration in sterile water
• Dry overnight under a Biological Safety Cabinet
• Dried plasmids can then be conserved at 4°C or room temperature, away from moisture for several weeks

PLASMIDS REHYDRATION

Materials

• ~3500ng dried plasmids prepared as described in the “Plasmid drying” protocol
• Sterile water

Methods

• Rehydrate plasmids in 15μL sterile water
• Flush for several minutes and incubate at room temperature for 10 minutes or more, to ensure maximal resuspension of DNA
• Flush again and you can then use plasmids as is they were conserved frozen

NB: YOU MUST ADD AN INCUBATION STEP IN ICE FOR AT LEAST 15 MINUTES IF YOU WANT TO USE THESE PLASMIDS FOR HEAT-SHOCK TRANSFORMATION USING BACTERIA MADE COMPETENT WITH CaCl2

RESTRICTION ENZYMES DRYING

Materials

• Commercial restriction enzymes (10X) and corresponding digestion buffer
• 0.5M trehalose solution
• Sterile water

Methods

PROTOCOL FOR A 15μL DIGESTION MIX WITH A FINAL CONCENTRATION OF 0.3M TREHALOSE, MADE TO DIGEST 1μg DNA AFTER RESUSPENSION

• Make aliquots containing 1μL enzymes, 1.5μL digestion buffer, 9μL 0.3M trehalose and 3.5μL sterile water
• Let dry under a Biological Safety Cabinet overnight
• Conserve dried enzymes at room temperature, or preferably in a 4°C fridge, away from moisture

RESTRICTION ENZYMES REHYDRATION

Materials

• Dried digestion mix, as described in the “Restriction enzymes drying” protocol (lien)
• Sterile water

Methods

NB: DIGESTION MIX MUST BE RESUSPENDED WITH A FINAL VOLUME OF 15μL FOR THE DIGESTION OF 1μg DNA

• Calculate the volume (V) of DNA solution you need to have 1μg DNA for digestion
• Calculate x=15μL-V, the volume of sterile water you need to resuspend the digestion mix
• Resuspend aliquots of digestion mix with the volume x of sterile water
• Flush for several minutes and incubate more than 10 minutes at room temperature
• Flush again and you can then use the entire aliquot to digest the volume V corresponding to 1μg DNA: the digestion sample final volume with enzymes and DNA is 15μL

FREEZE-DRIED BACTERIA RESUSPENSION SOLUTION

FOR 2mL RESUSPENSION SOLUTION (sterile water, DMSO 7%)

Materials

• 140μL DMSO 100%
• 1.860mL sterile water

Methods

• Add 140μL DMSO 100% per 1.860mL sterile water
• Homogenize by flushing
• Filter with a 22mm membrane filter
• Conserve at 4°C in a 2mL aliquot

CARBOHYDRATES (SUCROSE AND THREHALOSE) SOLUTIONS

FOR 10mL 0.5M SUCROSE SOLUTION (sterile water, sucrose 0.5M)

Materials

• 1.71g Sucrose 100%
• Sterile water to 10mL

Methods

• Add 1.71g Sucrose 100% in sterile water to 10mL
• Homogenize by vortexing
• Filter with a 22mm membrane filter
• Conserve at 4°C in a 10mL aliquot

FOR 10mL 0.5M TREHALOSE SOLUTION (sterile water, sucrose 0.5M)

Materials

• 1.71g Trehalose 100%
• Sterile water to 10mL

Methods

• Add 1.71g Trehalose 100% in sterile water to 10mL
• Homogenize by vortexing
• Filter with a 22mm membrane filter
• Conserve at 4°C in a 10mL aliquot

Bacterial transformation on petri dishes for DH5α

Materials (x8)

• 25µl DH5α competent cells
• DNA (concentration > 120ng/µl)
• 450 µl SOC
• antibiotic chloramphenicol (1000x)
• petri dishes

Methods

NB : work under microbiological safety bench and on ice
1. Prepare two sets of 4 Eppendorf tubes 2. Add DNA with a concentration of 120, 12, 1.2 and 0.2 ng/µl in 25ul of competent bacteria. 3. Gently invert the tubes 4-5 times to mix cells and DNA. Do not vortex 4. Incubate 30minutes on ice the first set and 30minutes in the cooling module, the second set 5. Heat shock at 42°C for 1minute in a dry bath for the first set and in the heating module for the second set. Do not mix 6. Place on ice for 2minutes the first set and in the cooling module for the second set. Do not mix 7. Add 450uL of SOC 8. Incubate at 37°C and 200rpm for 1h, add 1 µl of antibiotic, wait for 1h 9. Mix the cells thoroughly by inverting the tube 10. Deposit 50ul on the petri dish 11. Incubate overnight at 37°C with dishes upside down

Bacterial transformation on petri dishes for Top10

Materials (x8)

• 25µl Top10 competent cells • DNA (concentration around 50 ng/µl) • 450 µl SOC • antibiotic chloramphenicol (1000x) • petri dishes

Methods

NB : work under microbiological safety bench and on ice

• Prepare two sets of 2 Eppendorf tubes
• Add DNA in 25ul of competent bacteria.
• Gently invert the tubes 4-5 times to mix cells and DNA. Do not vortex
• Incubate 30minutes on ice the first set and 30minutes in the cooling module for the second set
• Heat shock at 42°C for 1minute in a dry bath for the first set and in the heating module for the second set. Do not mix
• Place on ice for 2minutes the first set and in the cooling module for the second set. Do not mix
• Add 450uL of SOC
• Incubate at 37°C and 200rpm for 1h, add 1 µl of antibiotic, wait for 1h
• Mix the cells thoroughly by inverting the tube
• Deposit 50ul on the petri dish
• Incubate overnight at 37°C with dishes upside down

Bacterial transformation

Materials

• 25µl DH5α competent cells
• 25 µl Top10 competent cells
• DNA (concentration around 50ng/µl)
• 450 µl SOC
• antibiotic chloramphenicol (1000x)

Methods

NB : work under microbiological safety bench and on ice

• Add DNA in 25ul of competent bacteria for each bacteria (Top10 and DH5α).
• Gently invert the tubes 4-5 times to mix cells and DNA. Do not vortex
• Incubate 30minutes in the cooling module
• Heat shock at 42°C for 1minute in the heating module. Do not mix
• Place inside the cooling module for 2minutes. Do not mix
• Add 450uL of SOC
• Incubate at 37°C and 200rpm for 2h,wait for 1h, add the antibiotic, wait for 1h

Fluorescence

• Power on the microplate reader and set to 37°C the inside temperature
• Add 200 µl of each tube in a well of a microplate and measure the OD and fluorescence every hour.