Team:Montpellier/Protocols

Protocols

Lactobacillus jensenii protocols

Materials : for 10 tubes of 50µL of competent cells

Glycerol stock or fresh culture of L. jensenii
MRS agar plates with appropriate antibiotic
100mL of liquid MRS
25mL of liquid MRS 80mM MgCl₂ (for recovery)
25mL of cold wash solution (10mM MgCl₂)
25mL of cold electroporation buffer (0.5M Sucrose, 10% glycerol)
Cold 1.5mL Eppendorf tubes
Cold electroporation cuvettes
Culture tubes for recovery

Protocol : preparation of competent cells

Grow cells overnight (ON) in MRS at 37°C
Use ON culture to inoculate 100mL MRS
Incubate at 37°C until OD₆₀₀ = 0.4 to 0.6 (about 3 hours)
Split the 100mL culture in two 50mL falcon tubes
Centrifuge for 5min at 3000g at 4°C
Starting from here, do everything on ice
Wash 2x with cold Wash Solution (WS) :

resuspend the pellet in each tube with 5mL of WS
centrifuge for 5min at 3000g at 4°C
repeat

Wash 1x with cold Electroporation Buffer (EB) : resuspend the pellet in each tube with 5mL of EB
Centrifuge for 5min at 3000g at 4°C
Resuspend each pellet in 5mL of EB
Concentrate in EB :

centrifuge for 5min at 3000g at 4°C
resuspend each pellet in 250µL and transfer these to 1.5mL Eppendorf tubes
make 50µL aliquots

Add about 1µg of plasmid DNA
Keep aliquots up to 2 hours

Protocol : electroporation of competent cells

Fill the cuvettes with the samples without making bubbles
Electroporate at 12.5kV/cm, 25µF, 600𝛀 (time constants for this protocol are between 11ms and 13ms)
Recover cells by adding 1mL of 80mM MgCl₂ MRS
Incubate ON at 37°C
Plate to select

Bacillus subtilis protocols

Materials : for 22 tubes of B. subtilis competent cells

Glycerol stock of B. subtilis
1x LB or LB + ATB plate
2x 50 or 100mL shakeflask
Medium A
Medium B
1.5mL tubes
1mL, 10mL and 25mL pipettes
Incubator equipped with shaker and set at 37°C
Spectrophotometer cuvettes
Spectrophotometer for OD₆₅₀nm determinations

Culture media to prepare :

10X Medium A base

Yeast extract : 10g
Casamino acids : 2g
Distilled water : fill to 900mL
Autoclave, then add :
50% glucose, filter-sterilized : 100mL
Aliquot by 10mL
Stock at RT

10X Bacillus subtilis salts

(NH₄)₂SO₄ 20g
K₂HPO₄ 3H₂O 183g
KH₂PO₄ 60g
Na⁺ citrate 10g
MgSO₄ 7H₂O 2g
Distilled water : fill to 1000mL
Aliquot by 9mL
Stock at RT

50mM CaCl₂ 2H₂O

Filtered 0.2µL
Stock at RT in 50mL tubes

250mM MgCl₂ 6H₂O

Filtered 0.2µL
Stock at RT in 50mL tubes

Medium A (200mL)

Sterile H₂O 162mL
10X Medium A base 20mL
Bacillus salts 18mL
Mix
Filter at 0.2µm (filtration unit)
Stock at RT

Medium B (100mL)

Medium A 100mL
50mM CaCl₂ 2H₂O
250mM MgCl₂ 6H₂O
Mix
Filter at 0.2µm (filtration unit)
Stock at RT

Protocol :

Day 1 :

Streak the strain on a petri dish from the glycerol stock on a LB Agar plate or LB + antibiotic if the train contains a resistance gene
Incubate overnight (18h) at 37°C

Day 2 :

In a flask, inoculate 20mL of medium A with few colonies, until 0.2 < OD₆₅nm < 0.25 (approximately one line of colony, resuspend well before to measure the OD)
Incubate at 37°C with vigorous aeration and read the OD every 30 minutes for monitor
When the OD not increase anymore, incubate the culture 90 minutes more at 37°C
Dilute 10 times of this culture into some Medium B in a new flask (1mL of culture in 9mL of medium B)
Incubate at 37°C with vigorous aeration for 90minutes
Add 50% glycerol to the culture in a 1:10^th ration (1mL of glycerol in 10mL of culture)
Aliquot per 500µL in tubes of 1.5mL
Flash freeze tubes in liquid nitrogen
Stock tubes at -80°C

Materials :

Competent cells
Plasmid
Water bath at 37°C
Incubator at 37°C with shaking
Centrifuge
LB medium
LB + ATB plate

Protocol :

Defrost 2 tubes of competent B. subtilis cells (500µL/tube) in the water bath at 37°C
Add 500ng of plasmid in one tube (mix by inverting 2 or 3 times). Nothing to add in the other tube (there is the negative control)
Place the tubes 30 minutes in the incubator at 37°C with shaking
Add 200µL of LB per tube and incubate again for 30 min or more at 37°C with shaking
Centrifuge 2min at 4000 rpm at room temperarture
Remove 600μL of supernatant
Resuspend the pellet and plate on LB + ATB (for plating, we use glass beads, alternatively you can use a pipette rake)
Incubate inverted plates ON at 37°C

Dilute each colony in 100µL sterile ddH2O

Prepare the PCR Mix :

	1 reaction	5 reactions
Buffer B	5µL	25µL
Enhancer	5µL	25µL
Primer 1 (Sp 27 rev)	0.75µL	3.75µL
Primer 2 (P280)	0.75µL	3.75µL
dNTP	0.5µL	2.5µL
Kapa Polymerase	0.1µL	0.5µL
H₂O	12µL	60µL

In PCR tube : 1µL resuspended colony + 25µL PCR Mix

PCR Cycle : Kapa

Step	Temperature	Time
Initial denaturation	95°C	5min
	95°C	30s
30 Cycles	63°C temperature depend of primers	30s
	72°C	1min/2kb time according to the fragment size
	72°C	1min
	12°C	∞

Verify by gel the colony PCR : 0.8% agarose in TBE + 3µL Sybr Safe DNA
2.55µL PCR product + blue and 2µL ladder 1kb, 100V 30min (for a single gel)

Escherichia coli protocols

Steps :

Streak the E.coli from glycerol stock.
Pre-culture.
Culture and preparation of electrocompetent cells.
Transformation test.
Calculate the efficiency.

Materials : to prepare 2L of culture

E. coli NEB10β (NEB, C3020K), TG1. Stored at -80°C
pUC19 DNA (NEB N3041A): 50pg/µL in ddH2O. Stored at -20°C
Ampicillin (Sigma): 100 mg/mL stock solution in ddH2O, filter-sterilized. Stored at -20°C
Cam, Kana, Spec
SOC medium
2L LB medium
3L Pure H2O, autoclaved
2L of 10% (v/v) Glycerol in H2O, autoclaved (Measure 100mL of glycerol 99% in a measuring tube, add it in a 1L glass bottle, rinse the measuring tube with miliQ water, add the water in the same bottle, adjust the volume in the bottle to 1L with miliQ water)
2 x 250mL Measuring tubes autoclaved
1 x 500mL Measuring tubes autoclaved
4 x 500 ml centrifuge bottles (NALGENE)
PCR tubes or 1.5mL tubes
1mL syringes for Eppendorf pipette
10mL, 5mL and 2mL pipettes
1 x 15mL tube
High speed centrifuges (Sorvall)
Shaking incubator at 37°C
Electroporator (Bio-rad)
Electroporation cuvettes (Bio-rad, 0.1 or 0.2 cm)

Protocol :

Day 1 :

Streak the E.coli from glycerol stock (-80°C) onto a LB plate without antibiotic. Incubate the plate at 37°C overnight. The plate can be storage at 4°C in one week.

Day 2 :

Inoculate a single E.coli colony in 30 ml of LB medium. Vortex. Grow overnight at 250 rpm and 37°C. The amount of overnight culture depends on how many liters of competent cell you are going to prepare. For 1L culture you need 4mL of overnight culture.
Do a negative control of contamination with 4 antibiotics (Kam, Carb, Kana, Spec) : 2mL of preculture + 2μL Antibiotic (4 different tubes). Vortex. Grow overnight at 250 rpm and 37°C.
Prepare 2YT medium or LB medium (500mL in 2L flask). Keep them at RT°C.
Prepare the autoclaved 3L pure H2O, 2L 10% glycerol, 3 Measuring tubes, 4 centrifuge bottles, PCR tubes or autoclaved 1.5mL tubes, PCR racks, 10mL 5mL and 2mL pipettes, few 1mL syringes for Eppendorf pipette and 1 x 15mL tube. Afterward, keep them at 4°C.
Book the 37°C incubator for tomorrow (4 x 2L from 9 to 13 for example).
Book the centrifuge for tomorrow (Rotor F9 / 2500g / 4°C / during 3h).

Day 3 :

Cells preparation :

Check the negatives controls.
Transfer 4 mL of overnight culture into 2-liter flask containing 1L of LB medium (1:250) without antibiotic. Homogenize.
Do a negative control of contamination with 4 antibiotics (Kam, Carb, Kana, Spec): 2mL of culture + 2μL Antibiotic (4 different tubes). Vortex. Grow overnight at 250 rpm and 37°C.
For the culture in 4 x 2-liter flask (500mL/flask) with a sterilized 500mL measuring tube.
Incubate at 250 rpm and 37°C until the OD₆₀₀ is about 0.5 ~ 0.6 (~4h) (To measure the OD, do the blanc with 1mL LB medium).
Note : For NEB10β, it takes about 4 hours to reach OD 0.52. You should check the OD of your culture after 3 hours and 30 minutes incubation time.
After OD is reached, pour the culture into 4 pre-chilled 500 ml centrifuge bottles.
Balance the weight of the bottles with the culture and chill it on ice for 30 min.
Keep flasks as waste.
Note : From here on, everything should be kept on ice, in the cold room and done as rapidly as possible.
Cool down the centrifuge at 4°C.
Spin at 2500g for 20 min at 4°C.
Decant the sup in a “Flask –waste”. Be careful, always dry the pellet the most of possible and don’t touch the flask with the bottle.
Use a sterilized 250mL measuring tube. Re-suspend cell pellet by hand and quickly, don’t use vortex in 250mL/bottle of pre-chilled pure H2O. You can pool it in 2 bottles (2 x 500mL) equilibrate and then spin at 2300g for 10 min at 4°C.
Decant the sup in a “Flask – waste”.
Use the same sterilized 250mL measuring tube and re-suspend cell pellet as step 12, in 250mL/bottle of pre-chilled pure H2O, and then spin at 2300g for 10 min at 4°C.
Decant the sup in a “Flask – waste”.
Use a sterilized 250mL measuring tube and re-suspend cell pellet as step 12, in 250 mL/bottle of pre-chilled 10% glycerol, and then spin at 2300g for 10 min at 4°C.
Note : Take care when decanting because the cells lose adherence in 10% glycerol. Perform this right after centrifugation can reduce the lost.
Repeat step 16 for two times with 100mL/bottle of pre-chilled 10% glycerol.
18.Carefully decant the sup and re-suspend cells in the remaining volume (about 1 ~ 2 mL) with E.coli electrocompetent cells preparation and transformation with 2mL pipette. (If it’s not possible, add 500μL glycerol 10% and re-suspend the pellet.) Transfer it in the 15mL cold tube.
Note : This is best done by gently swirling rather than vortex. Or use plastic drop for gently flushing.
Measure the OD₆₀₀ of a 0, 5:100 dilution of the cell suspension. 5μL culture + 995μL H20, mix, mesure. The spectrophotometer can measure only until OD₆₀₀ = 1.0 (If the measure is too high, dilute it of a 1:2 dilution).
1.0 OD₆₀₀ = ~2.5 x 10⁸ cells/mL. For High efficiency we need OD₆₀₀ = 150–200.
Aliquot immediately by 80μL into 1.5mL tubes, drop into a bath of liquid nitrogen, and transfer to -80°C for storage.
For Gibson assembly we need OD₆₀₀ = 80.
Do a negative control of contamination with 4 antibiotics (Kam, Carb, Kana, Spec): Add 8mL LB medium in the 15mL Tubes with few μL of re-suspended pellet, Pour in 4 tubes (2mL/tube) + 2μL Antibiotic / tube. Vortex. Grow overnight at 250 rpm and 37°C.

Day 3 or 4 :

Cells transformation by electroporation and efficiency test :

Pre-warm the SOC medium at 37°C. Pre-warm the plate contain selection antibiotics in 37°C for 1 hour.
Pre-chilled cuvettes (Bio-rad, 0.1 or 0.2 cm) on ice for 5 minutes before adding the cell/DNA mixture.
Thaw the electrocompetent cells on ice. Add a) 100 pg of pUC19 control plasmid (2μL at 50pg/L) for efficiency test (it’s possible to do it with 1μL just take it in consideration when you do the efficiency calculation), or b) 5 μL of your DNA sample for experiments, or d) 1μL of Gibson Assembly product in 40 μl of the thawed competent cells, mix by gently pipetting, and transfer to the pre-chilled cuvette. Stand for 1 min.
Perform electroporation by Bio-Rad GenePluser, Ec1 for 0.1 cm cuvette (brown) and Ec2 for 0.2 cm cuvette(green). (EC1, Bacteria = 1.8Kv and 6ms for Blanc or without cell). Flush the cuvette immediately with 1mL 37°C pre-warmed SOC medium and transfer to a 12mL culture tube. Recover for 1h at 37°C, 200 rpm shaking.
Wash cuvettes with ethanol and after water two times.
For efficiency test :
Perform serial dilution. Dilute 10μL of the culture in 990μL LB, then plate 100μL on LB Ampi plate (= 10^-3).
Dilute 10μL of ^-3 in 990μL LB, then plate 100μL on LB Ampi plate (= 10^-5)
(pUC19 = Ampi resistant)
In experiment :
Plate 100μL on a plate. Centrifuge the culture, throw 800μL, and plate the pellet resuspend in the 100μL SN.
Incubate Over Night at 37°C.

Day 4 :

Calculate the competency in colonies.
Ex : On the 10^-3 plate we have 27 colonies = 2,7.10⁴ UFC/mL (in our tube after electroporation)
2μL pUC19 = 100pg = 2,7.10⁴ UFC
100ng = 2,7.10⁷ UFC
Efficiency 2,7.10⁸ UFC/μg pUC19

Trouble shooting :

Low colony number of pUC19 transformed plate :

Check the DNA quality and quantity. The OD 260:230 should be higher than 1.6 at least.
Check the preparation procedure of competent cells. Especially the cell concentration. The OD₆₀₀ of 100-fold dilution of competent cell should higher than 1 at least.
Check the cuvette. Rusted or additional water on its surface ?
Check the voltage and time constant. The typical time constant is 4.8 to 5.1. If the time constant is lower than 4.5 ms, check your DNA preparation procedure. You should elute your DNA by pre-warmed H2O.
Your sample volume should be less than 5λ in 50λ transformation reaction to prevent the dilution of competent cell concentration.

Materials : for chemocompetent cells

LB medium : For NEB 10 Beta = LB without antibiotic, for DH5 Alpha Z1 = LB Spectinomycin 50, for NEB Turbo = LB without antibiotic
TSS medium : To make 50 mL : 5g PEG 8000, 1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20), 2.5 mL DMSO and LB to 50 mL. Filter sterilized (0.22 μm filter).
LB plates
1.5mL tubes
50mL falcons
Ice
500mL flask for culture
Liquid nitrogen

Protocol :

Day - 1 :

Streak an LB plate (with ATB if needed) with the E. coli strain

Day 0 :

Inoculate 5mL of LB (with ATB if needed) with the strain from the fresh streak plate
Incubate overnight at 37°C

Day 1 :

Dilute the overnight culture into 50mL of LB without antibiotic at 1/500 (200μL in 100mL)
Incubated at 37°C until OD₆₅₀ reach 0.2-0.3 (3-4h)
Place 1.5mL tubes, racks, 10mL pipettes at -20°C
DO EVERYTHING ON ICE
Incubate the culture on ice for 10min in 50mL falcon tubes
Cool down the centrifuge to 4°C
Centrifuge the culture at 3000rpm 4°C for 10min
Remove the supernatant
Resuspend cells in 10% volume of TSS buffer
Aliquot cells in 100uL in 1.5mL tubes with multdistrib pipette
Force them with liquid nitrogen
Store at -80°C

To test them : use pUC19 as positive control and do not forget negative control.

Thaw gently 3 tubes of cells on ice
Add 1μL pUC19 in 1 tube, add nothing in the others (negative control) (keep cells on ice)
Incubate 30min on ice
Heat-shock cells at 42°C during 45s (in water bath)
Put back on ice after heat-shock
Add 900 μL pre-warmed SOC (37°C) (rich medium)
Incubate cells at 37°C with agitation during at least 30min
Centrifuge cells at 4000rpm during 2min, remove 800μL of supernatant
Plate the rest from pUC19 positive control on LB Carb plate
Plate 100μL from negative control respectively on LB Chloramphenicol, LB Kanamycin, LB Carbenicillin and LB Spectinomycin plates
Incubate at 37°C overnight

Efficiency should be around 10⁷ colonies/μg pUC19

Materials : for transformation of chemocompetent cells

Chemical competent cells
Ice
Clean DNA to transform
SOC
Selective agar plates

Protocol :

Thaw gently cells on ice
Add DNA (keep cells on ice)
Incubate 30min on ice
Heat-shock cells at 42°C during 45s (in water bath)
Put back on ice after heat-shock
Add 900 μL pre-warmed SOC (37°C) (rich medium)
Incubate cells at 37°C with agitation during at least 30min
Plate 100 μL of transformation in selective agar plate or centrifuge cells at 4000rpm during 1min, remove 800μL of supernatant and plate the rest
Incubate at 37°C overnight

Sperm analysis protocols

For each experience the spermatozoa were diluted to a 1/100 concentration. This dilution allows us to individually track spermatozoa in the video and avoid crowding whereas still having a significant number of spermatozoa. A total volume of 20µL of the sample is put on the slide. This volume was calculated to have a height of sample bigger that spermatozoa heads while reducing the proportion of cells in different focal planes

The microscope used was an Olympus IX71 with magnificence 20x ; 1000µs of exposure time. The camera used for the recording was a Phantom MIRO 320 vision research with 768x430 of resolution and 60 FPS. For each sample, 5 videos of 10 seconds were recorded.

Materials : for 100mL

NaCl 94.7mM
KCl 4.8mM
CaCl₂ 1.71mM
KH₂PO₄ 1.2mM
MgSO₄ 7H₂O 12mM
NaHCO₃ 25mM
Glucose 5.6mM
HEPES 20mM
Carbenicillin 1mL of 50mg/mL stock
Streptomycin 1mL of 100mg/mL stock
BSA 6g
ddH₂O

Protocol :

Use a 200mL bottle with 50mL of ddH₂O
Weigh and dissolve all powders in the water
Add the antibiotics
Fill with ddH₂O to 100mL
Store at 4°C

Materials :

Nisin 2.5% from Sigma
HCl 1M
NaCl powder
ddH₂O

Protocol :

Use 50mL tube with 10mL ddH₂O
Weigh and add 187,5 mg of NaCl in the ddH₂O
Add 0.5mL HCl 1M
Fill to 25mL with ddH₂O
Aliquot to 2.5mL in 10mL tubes
Store at 4°C
When you need to use nisin, add 100mg of nisin powder to 2.5mL eluent to have 400µg/mL nisin

Cloning by Gibson Assembly

Steps :

Design
PCR Q5
DpnI digestion
PCR Clean-up
Gibson Assembly
Transformation
Verifications
Storage

Materials and protocols :

PCR Q5 :

Materials :

Q5® Hot Start High-Fidelity DNA polymerase (NEB)
Primers 20μM
Template adjust to 1ng/μL
Ultrapure Water
PCR tubes
PCR machine
Agar gel material

Protocol :

Depending on the final quantity of fragment needed and PCR yield, you can perform either a PCR with 20μL, 40μL or several times 40μL. Mix 8μL Water + 10μL Q5 + 1μL Template + 0.5μL each Primer.

Step	Temperature	Time
Initial denaturation	98°C	30s
	98°C	10s
30 Cycles	50–72°C temperature depend of primers	20s
	72°C	30s/kb time according to the fragment size

Use of the NEB T_m Calculator is highly recommended.

Prepare a 0.8% agar gel for sample larger than 1kb and 1.5% for sample smaller than 1kb
Load 2.5μL of the PCR reaction with 0.5μL of loading dye in the gel and a 1kb ladder or 100bp ladder according to the size of your expected fragment
Image the gel

DpnI digestion :

Materials :

DpnI (NEB)
PCR machine

Protocol :

Add 1μL DpnI in 17.5μL tube from Q5
Mix well by pipetting up and down so that you solution is homogenous and glycerol is not in the bottom of the tube
1h at 37°C
10min at 80°C and hold at 12°C

PCR Clean-up :

Material :

Biosentec PCR Clean-up Kit

Protocol :

biosentec protocol
Measure the DNA concentration with Nanodrop

Gibson Assembly :

Materials :

2X Gibson Assembly Mix (see Gibson Assembly Mix protocol)
Insert fragment(s) from PCR or ordered
Vector fragment (after PCR, DpnI and clean-up)
Ultrapure water
Thermo-cycler at 50°C

Protocol :

Calculate the volume of insert and vector to mix in the Gibson Assembly reaction. For 2 fragment assembly (one insert and one vector), the optimum is to mix 100ng of vector with 3 times more insert in mole, and for more than 2 fragment assembly, 100ng of vector with same quantity of each insert in mole. The total volume of insert(s) and vector have to be 10μL or less, if the calculated total volume is higher, the quantity of vector can be reduced up to 50ng and the volume of insert calculated accordingly.
Mix the vector and insert(s) fragments according to previously calculated proportions with 10μL of the Gibson Assembly Mix and adjust the total volume of the reaction to 20μL with water. As negative control of assembly, mix the vector alone in the same proportion than previously with 10μL of Gibson Assembly Mix and adjust the total volume of the reaction to 20μL with water.
Place the reactions at 50°C during one hour

Note : For a better conservation of your Gibson Assembly reaction at room temperature you can incubate your reaction 10min at 80°C.

Transformation (not for L. jensenii) :

Colony PCR :

Materials :

2X One-taq quick load master mix (NEB)
Primers at 20μM
Colonies
PCR tubes
PCR machine
Agar gel material

Protocol :

For each cloning, perform two colony PCR from two different colonies
For each colony PCR, pick one colony and re-suspend it in 10μL of sterile water (in PCR tube)
Pre-mix the One-Taq master mix, primers and water for the corresponding number of reaction, such as for one reaction : 5µL of master mix, 0.25μL of each primer, 3.5μL of water
Keep the colony re-suspended in water at 4°C, to use afterward to inoculate the culture for plasmid extraction
Mix 9µL of the pre-mix with 1L of the re-suspended colony
Place the tube in the PCR machine with the following PCR cycle :
1. 95°C 5min
2. 95°C 20s
3. Temperature dependent on primers – 30s
4. 68°C – 1min/kb
5. Cycle 30 Times the 3 last steps
6. 68°C 5min
7. Hold at 12°C
Prepare a 0.8% agar gel for sample larger than 1kb and 1.5% for sample smaller than 1kb
Load directly 5μL of the PCR reaction in the gel and a 1kb ladder or 100bp ladder according to the size of your expected fragment
Image the gel

Plasmid extraction :

Materials :

LB with appropriate antibiotic
Falcon tubes
Incubator
Plasmid extraction kit from Qiagen

Protocol :

For the colony PCR with the corresponding fragment size, mix 5μL of the re-suspended colony in water in 5mL of LB for high copy plasmid or 10mL for low copy plasmid in a 50mL falcon
Place the culture at 37°C with agitation overnight
From the overnight culture, perform a strick of each culture in a petri dish with the appropriate antibiotic for further glycerol stock (Plate at 37°C overnight, and stored at 4°C)
Centrifuge the culture (5min at 5000rpm) and perform plasmid extraction according to protocol from Qiagen kit

Note : For low copy plasmid, double re-suspension, lysis and neutralization volume and elute in 30μL.

Sequencing and glycerol stock :

Materials :

GATC barcodes
Tubes 1.5mL
Primers
dd water
50% glycerol

Protocol :

Send the extracted plasmid DNA to sequencing with the appropriate primers to verify the full cloned sequence, follow GATC procedure (5μL DNA at 30 to 100ng/μL + 5μL of one Primer at 5μM)
For correct sequence, inoculate 2mL of LB with appropriate antibiotic with a colony from the corresponding streak
Place the culture at 37°C with agitation during 6 hours, until the culture is trouble
Mix 1.2mL of culture with 400μL of 50% glycerol (15% glycerol)
Annotate the tube (Name of construction / Strain / Antibiotic / Book ref / Date / Your name)
Place the glycerol stock at -80°C

Media, buffers and glycerol stock

Warm agar medium on microwave until agar have been dissolved (be careful it is hot! And open the bottle a little bit before to warm it). Warm time : 4 min / 200mL Bottle ; 6min + 5min + 3min / 700mL Bottle
Wait for the medium to be colder (around 50°C, when you can take it without burning yourself).
Add the antibiotic
Mix without making bubbles
Make plates
Let dry
Store them upside down, in a plastic bag, at 4°C. Identify the bag : Medium name, antibiotic name, your name, date

Materials :

Difco™ Lactobacilli MRS Broth powder
Agar
MiliQ H₂O

Protocol :

Weigh 13.7g of Difco™ Lactobacilli MRS Broth powder
Weigh 2.5g of Agar if you want to make agar plates
Add 250mL of miliQ H₂O in a 500mL bottle
Add powders to the bottle
Mix well until everything is disolved
Put it to autoclave
Store it at RT

Materials :

SOB medium powder
ddH2O
50mL graduated cylinder
Ten 100mL glass bottles
Glucose powder
Syringes
0.2µm filters
50mL sterile tube
5mL sterile pipettes
1.5mL sterile centrifuge tubes

Protocol :

Prepare SOB medium :

Dissolve 14g SOB medium into 500mL (final volume) ddH2O (28g/L final concentration).
Aliquot the solution with the graduated cylinder: 50mL in each 100mL glass bottle.
Autoclave them the same day and cool down.

Prepare 2M glucose solution :

Dissolve 18 g glucose into 50 ml (final volume) ddH2O and filter-sterilize into a sterile 50 mL tube under the hood PSM.
Aliquot 0.5mL per microcentrifuge tube under the hood PSM.
Store aliquots at -20°C.

Prepare SOC medium :

Add 0.5mL glucose solution into 50mL SOB medium before use.
Divide into 10mL aliquots with SOC medium to avoid contamination.

Materials :

Antibiotic powders (usually in the common fridge in the flow cytometer room)
ddH2O
100% ethanol solution (under the chemical hood)
Syringes
0.2μm filters
50mL tubes
1.5mL sterile centrifuge tubes
10mL sterile pipettes

Protocol :

Weight the powder.
Add ~50% of desired liquid to a 50mL tube.
Add powder to liquid in tube and mix to dissolve.
Adjust the volume to the desired final value.
Filter the solution, ONLY if the antibiotic is diluted in water, under the hood PSM.
Aliquot the solution under the hood PSM by 1mL in 1.5mL tubes using a 10mL pipette.
Store aliquots at -20°C.

Antibiotic	Stock concentration	Weight (x g in x mL)	Solution for dilution	Working concentration
Ampicillin	100mg/mL	5g/50mL	H₂O	100µg/mL
Carbenillin	50mg/mL	2.5g/50mL	H₂O	50µg/mL
Kanamycin	50mg/mL	2.5g/50mL	H₂O	50µg/mL
Spectinomycin	100mg/mL	1g/10mL	H₂O	100µg/mL
Chloramphenicol	25mg/mL	-	100% ethanol	25µg/mL
Neomycin	15mg/mL	750mg/50mL	H₂O	-
Streptomycin	100mg/mL	-	H₂O	-
Erythromycin	0.5mg/mL	0.025g/50mL	100% ethanol	-
Tetracyclin	10mg/mL	-	-	10µg/mL

Principle :

Freezing is an efficient way of storing bacteria. Glycerol allows to reduce the harmful effect of ice crystals of bacteria which can damage cells by dehydration caused by a localized increase in salt concentration leading to denaturation of proteins. Additionally, ice crystals can also puncture cellular membranes. Glycerol as a cryoprotectant depresses the freezing point of bacterial cells, enhancing supercooling. It does so by forming strong hydrogen bonds with water molecules, competing with water-water hydrogen bonding. This disrupts the crystal lattice formation of ice unless the temperature is significantly lowered. The final concentration have to be no more than 20%.

Materials : for cryogenic tubes

Cryogenic tube with screw cap volume 2mL
Overnight culture or 6h culture of bacteria in LB with antibiotic for selection
Sterile 50% glycerol
Cryo-box 100 positions

Protocol :

Label a cryogenic tube with sticker label : construction name, short name, plasmid, antibiotic resistance, strain, book reference, date and initials ...
In sterile conditions mix in a cryogenic tube : 300μL of 50% glycerol + 1200μL of culture.
Homogenize by inverting.
Store the tube at -80°C in a labeled plastic cryo-box 100 positions.

Materials :

PEG 8000
MgCl2*6H20
DMSO
LB medium
Syringes
0.2μm filters
15mL sterile tube

Protocol : to make 250mL TSS Buffer

Mix : 25g PEG 8000 + 1.5g MgCl2*6H20 in 12.5mL DMSO
Add LB to 250 mL.
Filter sterilize (0.22 μm filter) under the hood PSM.
Aliquot in 15mL sterile tubes.

Materials :

1.5mL microtubes
PCR tubes

To prepare the 5X Isothermal solution (ISO 5X) :

Tris-HCl pH 7.5 solution 1M
PEG-8000
MgCl₂ solution 1M
DTT 1M
dNTP Mix 10mM
NAD 100mM
ddH₂O

To prepare the Gibson Assembly Mix 2X solution :

ISO 5X
T5 exonucelase (10 U/µL)
Taq DNA ligase (40 U/µL)
Phusion DNA polymerase (2 U/µL)
ddH₂O

Protocol :

5X isothermal reaction buffer : preparation for 10mL final solution

	10mL final	Final concentration
Tris-HCl pH 7.5	5mL	500mM
PEG-8000	2.5g	50%
Vortex
MgCl₂	500μL	50mM
DTT	500μL	50mM
dNTP Mix	1mL	1mM
NAD	500μL	5mM
ddH₂O	Qsp 10mL	-

The 5X isothermal reaction buffer has to be aliquot in 1mL, in 1.5mL tubes.

2X Gibson Assembly mix

	x2	x2
Final volume (µL)	1000	1500
ISO 5X (μL)	300.8	451.2
T5 exonucelase (10 U/μL)	0.6	0.9*
Taq DNA ligase (40 U/μL)	150.4	225.6
Phusion DNA polymerase (2 U/μL)	18.8	28.2
ddH₂O	529.4	794.1

The Gibson Assembly 2X mix has to be aliquot in 10μl in PCR tubes on a rack. Put the rack at -20°C. When it’s freeze put all tubes in a “tips” box.

* : Or do a dilution 1/10 of the solution 10 U/μL and add 9μL for 1500μL final.

Materials :

Centrifuge
10mM Tris-HCl pH 8.0
PRC tubes to aliquot the DNA resuspended

Protocol :

Upon receipt, briefly centrifuge the tube or plate and add without mixing nuclease-free 10mM Tris-HCl pH 8.0 to the desired concentration : in 96-well plate add 10μL buffer. We do not recommend resuspension in water
Wait for 3 to 4 hours at RT
Resuspend by pipetting and briefly centrifuge if needed
Prepare aliquots of the stock dilution and separate working aliquots to limit chances of contamination and to reduce the number of freeze/thaw cycles. Use working aliquots as soon as possible after preparation and minimize exposure to high temperatures. (Keep the mother plate in case you need it after)
For long-term storage (up to one year), freeze DNA at -20°C. For even longer storage, freeze DNA at -80°C

Cleaning procedures

Used beads should be placed in recipient with bleach before washing.

Put the beads (without the bleach) in a bucket.
Add new bleach and water to cover all of the beads, wait 30min for full decontamination.
Remove the bleach, place the beads in a clean bucket and add water. Mix beads and water to be sure that all beads have been in contact with the water.
Re-iterate the previous step.
Perform twice step 3 with deionized water instead of water.
Perform once step 3 with ethanol 70%.
Remove ethanol.
Clean your bench.
Dry beads using paper (place paper on your bench, smoothly put beads on it and use paper to dry them).
Place beads into small bottles (you can use the paper as a funnel).
Autoclave the bottles (you can just add autoclave tap and place them on kitchen table).

Materials :

Dirty Gene Pulser / MicroPulser Electroporation cuvettes
2 500mL beakers
1L of 70% ethanol

Protocol :

Arrange the cuvettes side by side in two beakers identified A and B.
Fill cuvettes with 70% ethanol.
Add other lying cuvettes and remove the caps on top.
Fill the beakers up to 500mL with ethanol. Warning : all cuvettes must be filled.
Cover the beakers of double aluminum foil.
Sonicate the beakers alternatively 15min A, 15min B, 15min A, 15min B, in the sonication bath.
Cool on the bench overnight.
Empty the ethanol from the beaker by opening minimally the aluminum foil (to avoid contamination).
Make 5 to 8 holes in the aluminum of each beakers with a syringe needle to create a ventilation.
Dry the cuvettes in their beakers in an oven at 65°C (2 days or more).
When the tanks and caps are dry, close them under sterile condition (flame or under the hood).

Clean recommandation :

96 wells plate should be clean just after usage (at least in the 48h). Afterward cells tend to precipitate and it starts to be trickier to well clean them. If 96 well plates are store for a long time before cleaning, it is important to carefully control the plate after cleaning. Do not hesitate to throw plate which are not perfectly clean (and white).

Protocol :

Throw the liquid culture from the plate in a bucket and add bleach to it. After 30min, throw the liquid to the sink and clean the bucket and the sink.
Place the plate to a bucket and add bleach mix to water to completely fill the plate. Wait 30min for complete decontamination of the plate. (do not wait several days, it obstructs the sink and the plate will turn yellow with time).
Remove the bleach.
Clean the plate with water, repeat this step twice. You can use the tap to add water specifically to each well. Remove as much as possible liquid from the plate between two rounds of cleaning by manually shaking the plate.
Clean the plate with deionized water, repeat this step twice.
Clean the plate with ethanol, repeat this step twice.
Remove liquid from the plate and let it dry on paper in the reverse orientation during at least one day (depending the external temperature and humidity percentage).
Control the quality of the cleaning, if the plate is not perfectly clean: throw it, if it is clean, place it on the shelf with it friends.