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Protocols
1) Medium and solution protocol
Way to use it | Compound name | Protocol |
---|---|---|
DNA migration | Agarose gel 1% | 1g/100ml of agarose into TAE 1X |
Supercompetant Escherichia Coli sp. cells | Tfb1 | 30mM KOAc, 0.05M MnCl2, 10mM CaCl2, Glycerol 15% |
Supercompetant Escherichia Coli sp. cells | Tfb2 | 0,01M MOPS (pH=7), 0.075M CaCl2, 0.01 KCl, Glycerol 15% |
SDS PAGE | Loading buffer 2X | 200 mM Tris HCl (pH = 6.8), 4% SDS, 0.02% Bromophenol blue, 20% glycerol, 2.5% βME |
Electrophoresis buffer (10X) ( pH= 8,3) | 0.025 M Tris, 0.19 M glycine, 0.1% SDS | |
Discolouring (200ml) | 100 ml ethanol 96%, 20 ml CH3COOH, 169 ml water | |
Coomassie Blue (1L) | 1.2 g Coomassie blue, 250 ml ethanol 95%, 80 ml acetique acide | |
SES 4X | 50% glycerol, 20 mM EDTA, 1% SDS, 0.05% Bromophenol blue, 0.05% xylene cyanol | |
Western Blot | Rouge Ponceau (100ml) stored in dark | 0.2 g Ponceau S, 3% tricloroacetique acide |
2) Molecular biology
A) Supercompetant Escherichia Coli cells (+/- 80 tubes of competent cells)
- Streak E.coli cells on an LB agar plate with or without antibiotics.
- 37°C, overnight.
- Inoculate 3-4 colonies in 5 mL LB media (+ antibiotics selection if necessary),
- 37°C, overnight.
- Dilute the preculture to 1/100 in 200mL LB final(+/- OD600 = 0,05)
- 37°C, 250 rpm, until OD600 = 0,5 (+/-2 hours).
- Place cells on ice for 20 minutes.
- Centrifuge cells in Sorval GSA rotor : 4°C, 10 minutes, 3500 rpm.
- Discard supernatant
- Cells must remain cold for the rest of the procedure (every steps in ice)
- Discard the supernatant and resuspend cells in cold Tfb1 (30mM KOAc, 0,05M MnCl2, 10mM CaCl2, Glycerol 15%).
- Centrifuge cells using Sorval RT6000B rotor : 4°C, 5 minutes, 3500 rpm.
- Discard the supernatant et resuspend cells (by pipetting) in cold Tfb2 (1/10 volume of Tfb1) (0,01M MOPS pH=7, 0,075M CaCl2, 0,01 KCl, Glycerol 15%).
- Incubate on ice for 15 minutes.
- Transfer 120μL of cells into (1,5mL) Ependorff tubes placed on ice.
- Cells stored at -80°C can be used for transformation for up to about 6 months.>
Additional steps :
- Check the competence of bacteria by transformation (following “E. coli sp. Transformation protocol)
B) E. coli sp. transformation protocol :
- 1 ng of DNA plasmid into a tube of super competent cells
- 20 minutes in ice
- Heat shock : 1 minute at 42°C
- 5 minutes in ice
- Adding LB medium to Vf = 1 mL
- Shake : 1h, 37°C
- Centrifuge : 4000 rpm, 4 minutes
- Streak on agar plate (different dilution can be made)
C) Digestion/ligation cloning
Plasmid and insert digestion :
- DNA (plasmid or insert) : 1μg
- Enzyme 1: 2%
- Enzyme 2: 2%
- 1X buffer
- 37°C, 3H (depend of restriction enzyme used)
- PCR Clean up
Ligation :
- T4 ligase 5%
- Buffer T4 1X
- Vector : 30 ng
- Insert : 100ng
- 1h, room temperature
D) SLIC cloning
Protocol optimised by Gauthier (GDP) (13/10/2020)
- Vector digestion during 1H : 2 µg
- Insert amplification : by PCR Q5 Mix pour 50 µL :
- Oligo1 : 2,5 µL
- Oligo2 : 2,5 µL
- dNTPs : 1 µL
- 5xBuffer : 10 µL
- GC Enhancer : 10 µL
- DNA matrice : 1 µL
- Eau : 22,5 µL
- Q5 polymérase : 0,5 µL
- 98°C : 30 sec
- 98°C : 10 sec
- Tm°C : 30 sec
- 72°C : 15 sec (pour 500 bp) go to #2 30 cycles
- 72°C : 30 sec
- PCR Clean Up of insert(s) and restricted vector(s)
- Assembly of the cloning (s) and the control IN PCR TUBES (250 µL or 500 µL) with :
- Incubate between 2 minutes 30 secondes and 3 minutes at room temperature
- Incubate 10 minutes in ice
- Adding 100 µL of competent cells and perform a classic transformation protocol.
- 1 h in ice
- 2 minutes at 42 °C
- 5 minutes in ice
- 5 minutes at room temperature, adding 900 µl 2YT
- 1 h at 37 °C
- Spread on medium + Ab, 100 µl and all the rest (centrifuge 5 min at 5000 rpm, remove 850 µl and resuspend with the remaining 50).
Cloning | Control | |
---|---|---|
Vector restricted | 0,03 pmola | 0,03 pmola |
Insert | 0,09 pmola | 0a |
NeB Buffer 2.1 | 1 µL | 1 µL |
T4 DNA polymérase | 0,2 µL | 0,2 µL |
H2O ppi | Qsp 10 µL | Qsp 10 µL |
E) Megapriming
Mix PCR:
- 2,5 µl 10X reaction buffer
- 0,5 µl dsDNA template
- 1 µl primer 1 (10 µM)
- 1 µl primer 2 (10 µM)
- 5 µl dNTP (2.5mM)
- qsp H2O 24 µl
- 1 µl Pfu Turbo DNA polymerase (ou Pfu Ultra II)
Program:
- 30min, 95°C
- 16 cycles
- 95°C, 30min
- 55°C, 1 minute
- 68°C, 1 to 2 minutes/kb of plasmid
- Then, following the transformation protocol
3) Biochemistry
A) Overproduction : optimized protocol (pBAD24 induction)
- Bacteria preculture in LB medium with antibiotics : All day, 37°C
- Back diluted 1/100 in new LB medium with antibiotics
- 37°C until OD600nm = 0.4
- Induced with [Arabinose] = 1%
- 28°C, overnight
B) Westernblot protocol
- Following the overproduction proteins
- Take 1UDO and adding 20µL of loading buffer 2X
- 10min, 95°C
- 2x SDS PAGE gel migration : +/- 45min, 200V in YZ buffer 1X
The second SDS PAGE gel can be revealed into Bleu coomassie coloration - Western Blot : Transfert (Blot), construction
- 2x Wathman (previously soaked in transfer buffer)
- Nitrocellulose (pre-soaked in transfer buffer)
- 1x Gel SDS PAGE
- 2 xWathman (previously soaked in transfer buffer)
- 30 to 45 min, 80V (depend of the protein size)
- Western Blot : Antibody revelation (following the company protocol)
- Blocking
- First antibody
- Washes
- Second antibody
- Revelation (depend of secondary antibody)
C) Purification protocol : strep tactin II column
- Strep-Cyt1Aa overproduction : Following the “Overproduction : optimized protocol (pBAD24 induction)” protocol. The protocol was done with 2 litters of LB medium.
- On morning : DO 600nm measurement
- Centrifuge cells : 5000g, 20min, 4°C.
- Resuspend the pellet with 10mL of PBS 1X
- Centrifuge cells : 5000g, 20min, 4°C.
- Discard the supernatant
Cells lysis :
- Resuspension of the pellets in 10 mL of buffer W (buffer for Strep-Tactin purification column)
- Adding lysozyme (1mg/mL)
- 37°C, 30min
- Adding Protease inhibitor 1X and Dnase 1 (0.1mg/ml)
- Adding MgCl2 (10mM)
- 20min, room temperature
- Destroy cells using french-press
- Ultracentrifugation : 45 000g, 1H
Proteins purification (using Strep-Tactin Purification column)
- For 1 liter of culture: put 1 ml of the bead mixture on the column
- Balance the balls: 3 passes of 5 volumes of column (CV) of buffer W
- Adding lysat (supernatant after ultracentrifugation) with a ball
- Incubate 1H, 4°C, on the balance
- Pass all of the lysate through the column
- Washes 5 fois, 5CV tampon W
- Elution :
- Adding 500µL of elution buffer
- Incubate 20min à 4°C
- Adding 1ml of elution buffer (repeat 2 times)
- Washing the column :
- 5-10mL of H2O
- 5mL NaOH 1mM
- 5ml H2O
- 5ml Ethanol 20%
- Keep the column in ethanol , conserved at 4°C
- Doing SDS PAGES and Western Blot analysis
D) Timer lysis device experimentation
- Test timers: objective 1 (To determine if mitomycinC has an effect -dose or effect on/off. Choice of the culture DO and dose of mitomycinC for subsequent tests).
- Preculture strains MG1655 pRL -GFPcolA, MG1655 pRL -lysRFP and MG pRL1 LB medium with antibiotics
- 37°C, overnight
- Back Dilution at OD 0.01, culture 25 mL at 37°C with agitation. (Two DOi to test: phase expo DO=0.4 and phase stat DO=1.)
- We OD600nm is 1 or 0.4 : culture into 5 erlens of 5 mL (i.e. 15 erlens).
- Induced gene : Addition of [mitomycin C]: 0, 50, 300, 500 and 800 ng/mL.
- Incubation at 37°C.
- Test timers: objective 2 (follow the” accumulation of PFM and PFR in the course of time).
- Preculture strains MG1655 pRL1, MG1655 pRL -GFPcolA and MG1655 pRL -lysRFP in LB medium with antibiotics
- 37°C, overnight
- Back Dilution to OD 0.01, culture 50 mL at 37°C with agitation. (Two DOi to test: phase expo DO=0.4 and phase stat DO=1.)
- At DOi expected, separate cultures into 2 erlens of 20 mL: an untreated control and a mitomycin C treated: [mitomycin C] = 800 ng/mL.
- Incubation at 37°C for 5 hours.
- Take : 200 µL sample : reading fluoGFP and flouGFP (reading with a plate 96 black wells at TECAN)
- Sample of 1 mL : centrified for 5 min at 8000 rpm. Sampling of SN for samples induced.
- Cap + 40 µL charging buffer. vortex. 5 min at 95°C and then ice.
- 1 mL SN + 10% TCA (trichloroacetic acid).
- 30 min on ice, centrifugation for 20 min, 14 000 rpm and remove SN
- add 200 µL acetone 100%.
- Centrifugation: 15 min 14 000rpm
- Take all the SN. Allow the pellet to dry in the oven at 37°C
- Resume in 40µL of charge buffer.
- SDS PAGE gel migration analysis : Cap uninduced / Cap induced/induced SN (for each sample)
Every 30 min for 5 hrs take 200 uL sample for measuring : DO600, fluoGFP and flouGFP (reading with a plate 96 black wells at TECAN)
Every 30 min.