Basic 6His purification with pET expression

Material

E. Coli strain (BL21)
Plasmids JS 321 (GFP)

Day -1

Transform BL21 strain with pET GFP on LB + Ampicillin at 37°C

Day 0

Make a starter at the end of the afternoon = inoculate 10 mL of LB + ampicillin (4µL/mL) with one colony that you pick with a toothpick
Grow at 37°C overnight

Day +1

Inoculate 100 mL of LB + 400 µL ampicillin with 1 mL of the preculture from the night in an erlen of 500 mL.
Incubate 1h ~ 37⁰C 180rpm → until OD= 0,6-0,8
Collect 1mL of non induced "NI" (before iPTG) in an eppendorf
Add 1mL of iPTG up to 1mM
Grow at 30°C during 3h with 180 rpm or until OD=3
Collect 1mL of induced "I" (after iPTG) in an eppendorf
If it’s too concentrated you dilute per 1/10 to check the DO
Divide into 2 falcons 50 mL
Centrifuge for 15 min at 4000 rpm
Throw away the supernatant and keep the pellet dry on the freezer

Day +2

Resuspend pellet in 15 mL of buffer 1
Add DNAse (0,1 mg/mL) & lysozyme (0,1 mg/mL) only for emulsiiflex C5
French press/ emulsiflex C5 for cell lysis
Spin down at 45000 rpm for 30 min in the SS34 rotor
Wash 500 µL (1ml total -> (50% beads on ethanol) of Nickel beads in an Econocolumn Biorad
Retrieve 1ml of our solution “before N beads” and put it on the side
Close the bottom of the column and transfer the supernatant on it. (retrieve it on a falcon “non retenu”)
Incubate 15 min at 4°C on a wheel
Wash with 15 mL of buffer 1 (retrieve on falcon)
Wash with 5 mL of buffer 2 (retrieve on falcon)
Wash with 10 mL of buffer 1.(retrieve on falcon)
Elute 5 times with 0,5 mL ( ou 100 µL) of buffer 3 in eppendorf tubes (filled with 0,156mL of glycérol at 25%)
Homogenize, aliquote (200µL), freeze in liquid nitrogen and store at -80°C

Analysis

0,2 uDO600 of on induced and induced total cells
Equivalent 0,5 uDO600 extract (avant les billes de nickels)
Equivalent 0,3 uDO600 unbound
5 µL wash 1,2 and 3
2 µL of E1, E2, E3, E4 and E5

Buffers

Buffer 1	Buffer 2	Buffer 3
20mM Tris-HCl pH 8.0	20mM Tris-HCl pH 8.0	20mM Tris-HCl pH 8.0
10mM imidazole	10mM imidazole	200mM imidazole
200mM NaCl	1M NaCl	200mM NaCl
2mM 𝝱-mercaptoethanol	2mM 𝝱-mercaptoethanol	2mM 𝝱-mercaptoethanol
H20 to 100mL	H20 to 50mL	H20 to 10mL

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization

Same protocol as "Basic 6His purification with pET expression" but Expression was induced by 4 mM IPTG in the presence of 10 mM copper sulfate. After 4 h of post-induction at 37 °C and 120 rpm, shaking was stopped in order to provide microaerobic conditions.

Material

E. Coli strain (BL21)
Plasmids JS 321 (GFP)

Day -1

Transform BL21 strain with pET GFP on LB + Ampicillin at 37°C

Day 0

Make a starter at the end of the afternoon = inoculate 10 mL of LB + ampicillin (4µL/mL) with one colony that you pick with a toothpick
Grow at 37°C overnight

Day +1

Inoculate 100 mL of LB + 400 µL ampicillin with 1 mL of the preculture from the night in an erlen of 500 mL.
Collect 1mL of non induced "NI" (before iPTG) in an eppendorf
Expression was induced by 4 mM IPTG in the presence of 10 mM copper sulfate. After 4 h of post-induction at 37 °C and 120 rpm, shaking was stopped in order to provide microaerobic conditions.
Collect 1mL of induced "I" (after iPTG) in an eppendorf
If it’s too concentrated you dilute per 1/10 to check the DO
Divide into 2 falcons 50 mL
Centrifuge for 15 min at 4000 rpm
Throw away the supernatant and keep the pellet dry on the freezer

Day +2

Resuspend pellet in 15 mL of buffer 1
Add DNAse (0,1 mg/mL) & lysozyme (0,1 mg/mL) only for emulsiiflex C5
French press/ emulsiflex C5 for cell lysis
Spin down at 45000 rpm for 30 min in the SS34 rotor
Wash 500 µL (1ml total -> (50% beads on ethanol) of Nickel beads in an Econocolumn Biorad
Retrieve 1ml of our solution “before N beads” and put it on the side
Close the bottom of the column and transfer the supernatant on it. (retrieve it on a falcon “non retenu”)
Incubate 15 min at 4°C on a wheel
Wash with 15 mL of buffer 1 (retrieve on falcon)
Wash with 5 mL of buffer 2 (retrieve on falcon)
Wash with 10 mL of buffer 1.(retrieve on falcon)
Elute 5 times with 0,5 mL ( ou 100 µL) of buffer 3 in eppendorf tubes (filled with 0,156mL of glycérol at 25%)
Homogenize, aliquote (200µL), freeze in liquid nitrogen and store at -80°C

Analysis

0,2 uDO600 of on induced and induced total cells
Equivalent 0,5 uDO600 extract (avant les billes de nickels)
Equivalent 0,3 uDO600 unbound
5 µL wash 1,2 and 3
2 µL of E1, E2, E3, E4 and E5

Buffers

Buffer 1	Buffer 2	Buffer 3
20mM Tris-HCl pH 8.0	20mM Tris-HCl pH 8.0	20mM Tris-HCl pH 8.0
10mM imidazole	10mM imidazole	200mM imidazole
200mM NaCl	1M NaCl	200mM NaCl
2mM 𝝱-mercaptoethanol	2mM 𝝱-mercaptoethanol	2mM 𝝱-mercaptoethanol
H20 to 100mL	H20 to 50mL	H20 to 10mL

DH5alpha/BL21 competent cells

Preparation

Spread 100 µL on a LB petri dish and let grow overnight at 37°C
Pick one isolated colony and make an overnight starter of 5 mL of LB in a 50 mL Erlenmeyer
The next day, recover your starters and seed 1/100 = 0,05 mL = 50 µL bacterial cells in 200 mL of LB in a 1000mL erlen, then incubate 2-3h at 37°C and 180 rpm → until OD600=0,4-0,6
Recover your cells and pool them in cold 50 mL Falcon tubes
Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and pipette tips at -80°C for future use
Centrifuge tubes at 4000 rpm and 4°C for 10 minutes
Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes
Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2
Take 100 μL aliquots in Eppendorf tubes and stock them at -80°C

Buffer 1

Product	For 40 mL
KAc (1 M)	1.2 mL
MnCl2 (0.5 M)	4 mL
KCl (1 M)	4 mL
CaCl2 (0.1 M)	4 mL
Glycerol (80 %)	7.5 mL
H2O (sterile)	19.3 mL

Buffer 2

Product	For 8.5 mL
KCl (1 M)	80 µL
MOPS (1 M)	400 µl
CaCl2 (0.1 M)	6 mL
Glycerol (80 %)	1.5 mL
H2O (sterile)	500 µL

Acrylamide Gel

Preparation

Clean up the glass plates (1mm) with water only and wipe them down
Use ethanol to remove residues of acrylamide
Make the acrylamide assembly and verify first that the thin plate is smooth at the bottom to avoid gel leaks
Fill the space between the 2 plates with water to the end using a wash bottle and check after a moment if the water flowed a lot or not (if it flowed a little bit it’s okay your assembly is up to be used)
Remove the totality of water using a wipe
Meanwhile, prepare the reactifs of acrylamide gel
Follow the dosages of the first gel and fill it in the space between the 2 plates and leave a space of 1 cm
Fill the rest of space with water for few minutes and then remove the water like before
Follow the dosages of the second gel and fill the rest of space
Insert the brush on the top and let it dry a few minutes
Put the acrylamide assembly in a box completed with water and put it in the fridge

LB Agar

Preparation

Heat bottle in microwave at 300 W for 22 min
Let it cool down until you can touch the bottle with your hand
Prepare and work in a sterilized environnement
Poor approximately 25 mL per petri dish (until the surface is recovered and If there's bubble you can use a flame to pop them)

If you need an antibiotic, see to the table below :

Antibiotic	color code	Volume to add in LB	Final [x]
Ampicilline 2 [x] : - 25 mg/mL - 100 mg/mL	4 blues	4 µL/mL	100 µg/mL
Kanamycine 2 [x] : - 10 mg/mL - 100 mg/mL	1 red	5 µL/mL	50 µg/mL
Tetracycline 15 mg/ mL	1 black	1 µL/mL	15 µg/mL
Chloramphenicol 30 mg/mL	2 green	1.6 µL/mL	50 µg/mL

Note : if you’re using different antibiotics you should divide your volume according to what you need in separate erlenmeyer.

Ligation

Preparation (20µL reaction)

T4 DNA ligase Reaction Buffer (10X) : 2 µL
Vector DNA (4kb) : 50 ng
Insert DNA (Akb) : 37,5 ng
H20 to 20 µL
T4 DNA ligase 1µL
Work on ice
Add the mixture in a microcentrifuge tube
To mix the reaction, pipe it up and down and microfuge briefly
For cohesive (sticky ends), incubate at room temperature for 10 min. → our case (for blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2h)
Heat inactivate at 65°C for 10 min
Chill on ice and transformation of the reaction into competent cells

Mini prep

Preparation (NEB Monarch miniprep kit)

Pellet 3 mL bacterial culture by centrifugation for 30s. Discard supernatant
Resuspend pellet in 200 𝞵L Plasmid Resuspension Buffer (B1) (pink)
Vortex ou pipet to ensure cells are completely resuspended. There should be no visible clumps
Lyse cells by adding 200 𝞵L Plasmid Lysis Buffer (B2) (blue/green). Invert the tube immediately and gently 5-6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex!
Incubate for 5 min
Neutralize the lysat by adding 400 𝞵L of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until the color is uniformly yellow and a precipitate form. Do not vortex!
Incubate for 2 minutes
Clarify the lysate by spinning for 10 minutes at 13000 ppm
Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through
Re-insert column in the collection tube and add 200 𝞵L of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifugate for 1 min. Discard flow-through
Add 400 𝞵L of Plasmid Wash Buffer 2 and centrifugate for 1 min
Centrifugate for 1 min one more time
Transfer column to a clean 1,5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min before inserting it into the clean microfuge tube
Add 30 𝞵L Hot Water (deionised) (70°C) to the center of the matrix. Wait for 1 min, then spin for 1 min to elute DNA

PCR on colonies

Context

For each colony

Prick a colony with a toothpick then screen it to make sure we don’t lose any (use a grid pattern with numbers adapted to the petri dishes)
Work in a sterile environment
Prepare 11 tubes: 9 colonies + 2 controls (+ and -)
1 PCR tube for verification = 13 µL
Do the preparation mix for 12 tubes to make sure we have enough
12*13 = 156 µL of mixture to be prepared (150 µL to make the calculation easier)
The master mix is 2X, it needs to be diluted 2 times (because we have a mixture for 50 µL final)
Put the mixture down below in each PCR tube :

Master mix	75 µL
Oligo T7	3 µL
Oligo T7 reverse	3 µL
Colony	1
H20 (150 µL)	75 µL

Start the PCR program with Tm = 55°C, elongation 1 min/kb

PCR

Preparation

Mix components:

	for 25 µL	for 50 µL
10 µL Forward Primer	1,25 µL	2,5 µL
10 µL Reverse Primer	1,25 µL	2,5 µL
Template DNA	Variable*	Variable
Master mix	12,5 µL	25 µL
H20	to 25µL	to 50µL

*We did use 0,5 µL on 13-06-2022

Assemble all reaction components on ice
Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin
Transfer reaction quickly to a preheated thermocycler (98°C)

Initial denaturation: 98°C, 30s
25-35 cycles:

98°C, 5-10 s
50-70°C, 10-30s
72°C, 20-30s/kb

Final extension: 72°C, 2 min
Hold: 4-10°C

Adherence

Preparation

Cut pieces of plastic: 9 x 9 mm (for PP, PE, PS)
Put it on a glass plate (becher)
Check the OD504 of the liquid containing the proteins
Add 1ml of liquid on the glass plates (A and B) (ex: 100μL E2 + 900 Buffer 3, et 400μL E2 + 600μL Buffer 3)
Put them on a rocking stirrer
Plate A → After 30 mins (short term analysis) , retrieve the liquide and check the OD504 → place the tape in a fluorometry machine tank and add 1 mL of deionized water (the same used to make the «white» on the machine), and check the OD504 once more
Plate B → After 3 hours (long term analysis) , retrieve the liquide and check the OD504 → place the tape in a fluorometry machine tank and add 1 mL of deionized water (the same used to make the «white» on the machine), and check the OD504 once more
Repeat those steps for every peptides and plastics you have
To analyse, calculate from the OD504 you had at the beginning, every protein loss (on the washes, and the liquide you retrieved) by substracted them
Use your plastic and deionized water on your tank to act as a «white» when testing the plastic
Use, a solution with different concentration, to know to what extend it’ll help the adherence
To do so, check the OD600 on a sectrophotometer (→ it will be too high for the fluoro)
To modify the concentration, use the buffer you previously used during the purif.

Note

Do the same, with only GFP on the plastic (to know if the GFP has an adherence property)

Transformation

Preparation

Thaw the cell tubes on ice (cells/ bacterias = 100 µL)
Recover the needed number of competent cell tubes, adding an additional negative control tube
Add DNA (1 µL) into the competent cell tubes
Incubate tubes on ice for 20 min
Incubate tubes at 42°C for 1 minute for thermal choc
Leave tubes on ice for 5 min
Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and mix (850 rpm)
Centrifuge tubes 4 min and 4000 rpm at room temperature. And get rid of 900 µL of supernatant
Resuspend and spread cells (100 µL) on Petri dishes with the desired antibiotic and incubate overnight at 37°C

Transformation	20 µL DH5α + 1 µL JS228	Ampicillin
Transformation	20 µL BL21 + 1 µL JS321	Ampicillin
Test	50 µL DH5α + 3 µL plasmid (100 pg/ µL)	Chloramphenicol
Transformation	1 tube of DH5α + 1 µL JS228	Ampicillin
Transformation	1 tube BL21 + 1 µL JS321	Ampicillin
Test	1 tube DH5α + 3 µL plasmid (100 pg/ µL)	Chloramphenicol

Note

Spread with balls when you have a little quantity of DNA = picogramme, or when you want to do a cloning to have isolated colonies
Spreading using streak spreading when you transform a plasmid in cells to avoid a bacterial mat

Laccase activities

Laccase activity was assayed using 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as substrate. Oxidation of ABTS was detected by following the absorbance at 420 nm (e420 nm =36000 M−1·cm−1) during 2 min with a spectrophotometer (Cary 60, Agilent Technologies, USA). The reaction mixture (1 mL) contained 10 µL of appropriately diluted enzyme sample, 890 µL of acetate buffer (100 mM pH 5.7) and 100 µL of 50mM ABTS in Milli Q® Water at 30°C. The ABTS was added to initiate the reaction. One unit (U) of laccase was defined as one micromole of substrate oxidized per minute in these described conditions.

Preparation

First, soak the filter paper in ABTS solution (2 mM ABTS, 100 mM phosphate-citrate buffer, pH 4)
Dry the paper at 60°C and add 10 µl of enzymes glycerol preps
Incubate at 30°C for 10 min

In addition, a 96-well plate assay was used to test the activity of the laccases. The experiments were done in two different pHs, pH 5 and pH 7, of phosphate-citrate buffer (100 mM). Absorbances were measured at A420 in one minute intervals with the Tecan M200 plate reader.

Phage Display

Phage Helper preparation

Day 1

Thaw TG1 cells at 37°C : dilute cells in 100ml 2YT prewarmed at 37°C, shaking 240 rpm and are grow to OD600 0.5
Add 8x1011 phages M13K07 helper phage in each → 30 min RT no shaking
Centrifuge 10 min at 3500g
Resuspend pellet in 300 ml 2YT + 50 µg/ml Kanamycin → 30°C ON shaking 250 rpm

Day 2

Centrifuge culture 30 min at 12000g 15°C (sterilized falcon)
Precipitate the phages in supernatant by adding 1/5 volume of (20% PEG8000 + 2.5M NaCl filtered) → 1h on ice
Centrifuge 15 min at 3000g 4°C
Resuspend phage pellets into 20 ml PBS sterile
Add 4 ml 20 % PEG8000, 2.5 M NaCl → 1h on ice
Centrifuge 15 min at 3000g 4°C
Resuspend pool phage pellet into 2 mL PBS (or more if 2 mL are not enough) (sterilized eppendorf)
Centrifuge 5 min at 15000g 4°C, put SN in fresh sterilized tube on ice
Centrifuge 5 min at 15000g 4°C, put SN in fresh tube on ice
Determine the phage titer : 1 OD260 = 3x1010 pfu/mL (lecture au Nanodrop, mode ADN)
Make several aliquots of 500 µL and stored them at -20°C

Phage’s bank

Day 1

3x109 TG1/pHEN4 cells in 100 ml 2YT prewarmed at 37°C + 100 µg/mL Ampicilline + 2 % glucose → shaking 240 rpm and are grow to OD600 0.5
Add 5x1011 phages M13K07 helper phage in each → 30 min RT no shaking
Centrifuge 10 min at 3500g
Resuspend pellet in 300 ml 2XTY + 50 µg/ml Kanamycin+ 100 µg/mL Ampicilline, 30°C ON shaking 250rpm

Day 2

Centrifuge cultures 30 min at 12000g
Precipitate the phages in supernatant by adding 1/5 volume of 20 % PEG8000 + 2.5 M NaCl → 1h on ice
Centrifuge 15 min at 3000g 4°C
Resuspend phage pellets into 20 ml PBS cold
Add 4 ml 20 % PEG8000, 2.5 M NaCl → 30 min on ice
Centrifuge 15 min at 3000g 4°C
Resuspend pool phage pellet into 2 ml PBS cold (sterilized Eppendorf)
Centrifuge 5 min at 15000g put in fresh sterilized tubes on ice
Centrifuge 5 min at 15000g 4°C → put SN in fresh tube on ice
Determine the phage titer : 1 OD260 = 3x1010 pfu/ml and put them at 4°C
Coat 5 wells of a Nunc immunoplate MaxisorbTM surface with 100 µl coating buffer PBS and 5 wells with 100 µl of 100 µg/ml of antigen in coating buffer PBS at 4°C ON

Selection 1

Day 1

Place Polystyrene plastic and polyethylene plastic in a isopropanol solution (90 %) 30 min RT (don’t forget the negative controls without the plastic and with PBS solution instead)
Culture of TG1 cells (8 μL) in 10 mL 2YT (in sterilized falcon) + 10 mL 2YT as a control
Discard the isopropanol solution + rinse 4 times with 2mL PBS 0.05 % tween 20 and 4 times with 2 mL PBS
Block with 1mL PBS 2 % BSA/well → 2h RT (1h can be enough)
In the same time block 1mL phages in 1 mL PBS-BSA 2 % → 2h RT (1h can be enough)
Discard blocking solution + rinse 4 times the plate with 2 mL PBS 0.05 % tween 20 and 4 times with 2 mL PBS
Add 1 mL of blocked phages in each well
Keep at room temperature 2h (after 2h put le plate at 4°C until the TG1 DO600 = 0,25)
If TG1 DO600 > 0,25 dilute them to DO600 0,25
Rinse 15 times the plate with 2 mL PBS 0.05 % tween 20 and 4 times with 2 mL PBS
Elute the phages by adding 1 mL/well of 2 % BSA 0,2 M glycine HCl pH 2,5 → incubate for 10 min at RT
Pool the 5 wells and neutralize with 1 mL 1 M Tris pH 7.2 (pipetting several times to pick up the phages)

/!\From here, do the titration and the rest of the protocol at the same time

Add 4 mL of TG1 cells at OD600 0.5 to the remaining 2 mL of phages (Ag well only) → 37°C 30 min no shaking (in the same time with the titration test)
Add 18 mL 2YT + 100 μg/ml Amp + 2 % glucose → 37°C 15 min with shaking
Add 1.6x10^10 M13K07 helper phage stock → RT 30min no shaking
Centrifuge the cells at 4000 rpm for 15 min 4°C
Resuspend the pellet in 300 mL 2YT + 100 μg/mL Ampicillin + 50 μL kanamycin → 30°C ON 250 rpm

••••• Titer of panning and control •••••

Prepare dilutions 10-2 to 10-7 for Ag and 10-2 to 10-4 for control (10 µL Ag + 90 µL 2YT for 10-2 tube and 10 µL Ag + 90 µL of 10x-1 dilution tube for 10x tube)
Add 100µL of TG1 cells at OD600 0.5 to all the tubes → 37°C 30min no shaking

Plate 50µL on the small Petri according the following table :

Antigen		PBS (control)
Amp	Kana	Amp	Kana
10-2 to 10-7	10-2 to 10-4	10-2 to 10-4	10-2 to 10-4

Plate 50µL of TG1 on an Petri + Amp, Petri+kana and Petri without antibio (TG1 has no antibio resistance so they technically can throw on no-antibio Petri) → Incubate 37°C ON

Day 2

If the TG1 control is ok, estimate the titer and the enrichment factor and keep the 6 Petri + amp Ag at 4°C
Centrifuge cultures 30 min. at 11000g (distribute 50mL of culture per sterile falcon)
Precipitate the phages in supernatant by adding 1/5 volume of 20 % PEG8000 + 2.5 M NaCl 1h on ice (40 mL of supernatant + 8 mL of PEG+NaCl per new sterile falcon) → 1h on ice
Centrifuge 15 min 3000g 4°C
Resuspend phage pellets into final 20 ml of cold PBS
Add 4 ml 20 % PEG8000, 2.5 M NaCl → 30 min on ice
Centrifuge 15 min 3000g 4°C
Resuspend pool phage pellet into 2 ml PBS (in sterilized Eppendorf)
Centrifuge 5 min at 15000g, put in fresh sterilized tubes on ice
Centrifuge another time 5 min at 15000g, put in fresh sterilized tubes on ice and put SN in fresh sterilized tube on ice
Determine the phage titer; 1 OD260 = 3.1010 pfu/mL
Coat 5 wells of a Nunc immunoplate MaxisorbTM surface with 100 µl coating buffer PBS and 5 wells with 100 µl of 100 µg/ml of antigen in coating buffer PBS at 4°C ON

Selection 2

Same protocol as Selection 1 but with the phages from selection 1
If the titration gives good results (more than 3log difference between Ag and PBS control) it’s useless to precipitate the phages. You can do the Screening by ELISA
If it’s not, do an 3rd round of selection

ELISA screening

Day 1

Pre-culture 96 clones in a 96-well DW 🡪 1 mL 2YT (or LB) + Amp (100 µg/mL) ON 37°C 200rpm
Do not forget to start the culture of the HHV(s) for the + and NR controls

Day 2

EXPRESSION

Start cultures of x clones (12, 24, 48 depending on the project) from the pre-culture in DW 24 well: 4 mL 2YT + Amp (100 µg/mL) + 0.1 % glucose + 60 µL of pre-culture 🡪 37°C 200 rpm
Add 30 % glycerol to DW96, annotate (96 clones - antigen name - panning round number - date + DW map if required) and store at -80°C
When 0.5 < OD600 < 0.8 (about 2h) induce cultures with 1 mM IPTG (prepare a stock solution 2YT + Amp (100 µg/mL) + 10 mM IPTG and add 400µL per well for induction) 🡪 ON 28°C 200 rpm

ELISA

In a Nunc plate coater antigen (do not forget antigen for control(s)) and PBS (ctrl -): 100µL per well at 20µg/mL 🡪 ON 4°C

Day 3

TOTAL LYSIS

Centrifuge the DW 3000g 15 min 4°C
Empty the supernatant and freeze the pellets at -20°C minimum 1h
Thaw the pellets at RT for about 15 min ♦ Start washing the Nunc plate (ELISA) ♦
Add 100 µL of 20 µg/mL PBS+DNase solution per 1mL of culture to each well (i.e. 400 µL of solution for 4 mL) 🡪 Shake 250 rpm RT minimum 30 min (more convenient to do it in the infors)
Centrifuge 3000g 10 min 4°C
Dispense supernatants into the rinsed and blocked Nunc plate, respecting the plate layout

ELISA

Empty the plate and wash 3 times with 300 µL of PBS
Block the plate with 300 µL of 2 % PBS-milk 🡪 2h 37°C
Empty the plate and make 3 washes with 300 µL of PBS
Dispense the supernatants into the plate
Incubate ON at 4°C

Day 4

Empty the plate and make 3 washes 300µL PBS+Tween 0.1 % then 3 x 300 µL PBS
Add 100 µL/well of Ac-mouse-anti-HA solution 1/500 in PBS-milk2% 🡪 1h at 37°C
Empty the plate and wash 3 times 300 µL PBS+Tween 0.1 % then 3 x 300 µL PBS
Add 100 µL/well of 1/5000 Ac-anti-mouse-HRP solution in PBS-milk 1 % 🡪 1h at 37°C
Empty plate and wash 3 times 300 µL PBS+Tween 0.1 % then 3 x 300 µL PBS
Develop with either : BM Blue POD Substrate (ROCHE) 100µL/well and block the reaction with 50 µL of 1 M H2SO4
Or make the reagent yourself: dissolve 1 tablet of TMB (Tetramethyl benzidine Sigma T5525-50TAB) in 100 µL DMSO, add 9.9 mL 100mM NaAc pH6.0 + 2 µL H2O2 then block the reaction with 50 µL 1M H2SO4 (/!\ calculate the volume of reaction solution needed before and adjust the volumes)
Read the OD450nm

Killer digestion

Digestion mixture

5 microLitre ligation volume (empty pet + PP pet + PE pet)
1 microLitre of CutSmart Buffer
0.5 microLitre of Bag 1
3.5 microLitre H2O (QSP 10 microLitre)