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
Bacterial transformation
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
Bacteriophages protocols
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.
Solutions preparation for bacteriophages protocols
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.
DNA extraction (from lysis of bacteria by bacteriophages)
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.
Agarose gel electrophoresis
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
Protocol Gibson Assembly Master Mix
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.
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
Bacteria conservation
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
DNA conservation
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
Enzymes conservation
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
Preparation of the solutions for conservation
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
Protocols for the transformation module
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
Prepare two sets of 4 Eppendorf tubes
Add DNA with a concentration of 120, 12, 1.2 and 0.2 ng/µl 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, 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 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 and fluorescence kinetic
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
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.
