Difference between revisions of "Team:Brasil-USP/Notebook/protocols"
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<ul> | <ul> | ||
<li>On ice, prepare the following mixture in a microtube:</li> | <li>On ice, prepare the following mixture in a microtube:</li> | ||
| − | <ul>500 -1000 ng of plasmidial DNA</ul> | + | <ul>   - 500 -1000 ng of plasmidial DNA</ul> |
| − | <ul>1 μL of Restriction Enzyme 1</ul> | + | <ul>   - 1 μL of Restriction Enzyme 1</ul> |
| − | <ul>1 μL of Restriction Enzyme 2 (if necessary)</ul> | + | <ul>   - 1 μL of Restriction Enzyme 2 (if necessary)</ul> |
| − | <ul>2 μL of 10x FastDigest Buffer</ul> | + | <ul>   - 2 μL of 10x FastDigest Buffer</ul> |
| − | <ul>Nuclease free water to complete 20 μL</ul> | + | <ul>   - Nuclease free water to complete 20 μL</ul> |
<li>Spin the mixture.</li> | <li>Spin the mixture.</li> | ||
<li>Incubate at 37°C for at least 3 hours.</li> | <li>Incubate at 37°C for at least 3 hours.</li> | ||
Revision as of 14:01, 17 September 2015
Protocols
Notebook
Protocols
Table of contents
- Circuit Assembly Protocols
- Calcium chloride transformation with heat shock in Escherichia coli DH5α
- Plasmid extraction
- Digestion of plasmidial DNA
- Ligation reaction (Cohesive ends)
- Agarose Gel Electrophoresis
- Directed mutagenesis PCR (for restriction site elimination) using a plasmid template for restriction site elimination
- PCR amplification (applied to lcp)
- PCR amplification for difficult amplicons (applied to roxA)
- Gibson assembly
- Characterization Protocols
Calcium chloride transformation with heat shock in Escherichia coli DH5α
Materials
- Sterile LB agar plate supplemented with the appropriate antibiotic (ampicillin 100 μg ml-1 or chloramphenicol 34 μg ml-1 - SIGMA-ALDRICH®);
- Sterile liquid LB media (SIGMA-ALDRICH®);
- Competent DH5α cells (Novagen) prepared through heat shock with calcium chloride;
- Plasmidial DNA.
Methodology
- Put the 0.5 mL microtube containing 50 μL competent cells aliquot on ice;
- Add 20-50 ng of plasmidial DNA or 10 μL of ligation reaction to the competent cells. Mix by pipetting carefully;
- Place the tube into a 42°C water bath for 2 min;
- Return the tube to the ice for 5 min;
- Add 200 μL of liquid LB;
- Incubate at 37°C, 250 rpm for 45 min;
- Plate the liquid LB containing the bacterial suspension on a LB agar plate with the appropriate antibiotic;
- Incubate overnight (14- 16h) at 37°C.
Plasmid extraction
PureLink® Quick Plasmid Miniprep Kit-Life Technologies
Methodology
- Cell Growth
- Resuspension
- Lysis
- Neutralization
- Washing
- Elution of plasmidial DNA
- After isolating a single colony from a LB agar plate, grow it in 6 mL of liquid LB within the appropriate antibiotic. Incubate overnight (14-16h) at 37°C in a shaking incubator.
- Pellet the overnight culture in a 2 mL microtube and discard the supernatant. Repeat this step until the total liquid culture is finished. Resuspend the cell pellet in 240 μL of resuspension buffer by vortexing.
- Add 250 μL of the Lysis buffer. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this period.
- Add 350 μL of the neutralization solution. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this time. Centrifuge at 16000 g for 10 minutes.
- Transfer the supernatant to a new 1.5 mL microtube with the resin. Be careful not to transfer the white pellet.
Add 650 μL of Wash buffer. Centrifuge at 16000 g for 1 minute. Discard the supernatant. Centrifuge again for 2-4 min to remove ethanol remains.
- Put the resin in a new 1.5 mL microtube. Add 50 μL of nuclease free water at 65°C. Centrifuge at 16000 g for 3 minutes and discard the resin. Store DNA at -20°C.
Digestion of plasmidial DNA
Materials
- Plasmidial DNA;
- Restriction Enzyme 1: EcoRI or XbaI (FastDigest Thermo Scientific);
- Restriction Enzyme 2: SpeI or PstI (FastDigest Thermo Scientific);
- FastDigest Buffer (Thermo Scientific);
- Nuclease free water.
Methodology
- On ice, prepare the following mixture in a microtube:
- 500 -1000 ng of plasmidial DNA
- 1 μL of Restriction Enzyme 1
- 1 μL of Restriction Enzyme 2 (if necessary)
- 2 μL of 10x FastDigest Buffer
- Nuclease free water to complete 20 μL
- Spin the mixture.
- Incubate at 37°C for at least 3 hours.
- Perform agarose gel electrophoresis to confirm the results
- - 500 -1000 ng of plasmidial DNA
- - 1 μL of Restriction Enzyme 1
- - 1 μL of Restriction Enzyme 2 (if necessary)
- - 2 μL of 10x FastDigest Buffer
- - Nuclease free water to complete 20 μL
Ligation reaction (Cohesive ends)
ref: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202
Materials
- Vector DNA digested;
- Insert DNA digested;
- 10X T4 DNA Ligase Buffer* (Thermo Scientific);
- T4 DNA Ligase (Thermo Scientific);
- Nuclease free water.
- Set up the following reaction in a microcentrifuge tube on ice:
- For cohesive ends, incubate at 22°C for 3 hours + 16°C for 9 hours.
Methodology
Agarose Gel Electrophoresis
Materials
- 1X TAE Buffer;;
- Electrophoresis apparatus (cell, gasket, power supply, gel caster and comb; BIO-RAD - http://www.bio-rad.com/cmc_upload/Literature/38717/M1704400B.PDF);
- Gel analysis and documentation equipement (Gel DocTM EZ System, BIO-RAD);
- UV light box
- DNA ladder (Invitrogen or Thermo Scientific);
- 10X Loading Buffer (Invitrogen);
- Ethidium bromide (Promega);
- x% (mass/volume) agarose gel (the concentration varies with the size of the DNA sample; 1.2% is recommended to short DNA fragments - smaller than 200bp - and 0.8% is recommended to high length of DNA)
Methodology
- Agarose gel: Mixture 1X TAE with agarose (1.2 or 0.8 grams for each 100ml of buffer depending of x% agarose) and melting the mixture. Add ethidium bromide and after, transfer the melted gel into a gasket in a gel caster support with an appropriate comb);
- Prepare samples by diluting in loading buffer to approximately 1X or higher;
- Load the DNA ladder into the first well of the gel and the samples into the additional wells;
- Transfer gel to a cell and apply DNA ladder and samples;
- Run the gel for about 40 minutes at 100 volts;
- Do analysis (in Gel Doc equipment) or cut the gel (UV light box).
Directed mutagenesis PCR (for restriction site elimination) using a plasmid template for restriction site elimination
Materials
- 1 μl of 10 ng/μl DNA template;
- 5 μl of each primer forward and reverse (diluted to 20μM) previously designed and purchased;
- 1 μl of dNTP mixture 10mM;
- 10 μl Phusion HF 5X buffer (NEB) with MgCl2;
- 1 μl High Fidelity enzyme (2.5U μl-1, NEB);
- Sterile deionized water to 50 μl.
Methodology
- In a thermal cycler (BIO-RAD) set the fellow steps:
First (1X): 95°C for 3 min;
Second (18X): 95°C for 30s; 60°C for 30s (primers Tm); 72°C for 5 min (15-30s per kb - pUC9::roxA : 4462 bp)
Third (1X): 72°C for 15min;
Hold in 4°C.
- Run a gel electrophoresis to analysis (10 μl) and purification (all remainder reaction);
- Prepare a DNA digestion with only DpnI enzyme (Thermo Scientific);
- Heat shock in E. coli DH5α with 10 μl of the digest reaction;
- Do minipreps with some colonies;
- Confirm the mutation with a digest reaction with two enzymes, one vector site containing and with the desired mutation site. Confirm with a gel electrophoresis.
Methodology
- In a thermal cycler (BIO-RAD) set the fellow steps:
- Run a gel electrophoresis to analysis (10 μl) and purification (all remainder reaction);
- Prepare a DNA digestion with only DpnI enzyme (Thermo Scientific);
- Heat shock in E. coli DH5α with 10 μl of the digest reaction;
- Do minipreps with some colonies;
- Confirm the mutation with a digest reaction with two enzymes, one vector site containing and with the desired mutation site. Confirm with a gel electrophoresis.
- First (1X): 95°C for 3 min;
- Second (18X): 95°C for 30s; 60°C for 30s (primers Tm); 72°C for 5 min (15-30s per kb - pUC9::roxA : 4462 bp)
- Third (1X): 72°C for 15min;
- Hold in 4°C.
PCR amplification (applied to lcp)
Materials
- 1μl DNA template at 10 ng μl-1;
- 5μl of each primers forward and reverse (diluted to 20μM) previously designed and purchased;
- 1μl of dNTP mixture 10mM;
- 5μl High Fidelity 10X buffer (Thermo scientific) with MgCl2;
- 2.5μl BSA protein (Promega);
- 0.5μl High fidelity enzyme (2.5 U μl-1, Thermo Scientific);
- Sterile deionized water quantum sufficit for 50 μl.
Methodology
- In a thermal cycler (BIO-RAD) set the following steps :
First (1X): 95°C for 3 min;
Second (30X): 95°C for 30s; 57°C for 30s; 72°C for 2 min (1-2 min per kb - Lcp : 1128 bp);
Third (1X): 72°C for 10min;
Hold in 4°C.
- Run a gel electrophoresis to analysis (3 μl) and purification (all remainder reaction).
- First (1X): 95°C for 3 min;
- Second (30X): 95°C for 30s; 57°C for 30s; 72°C for 2 min (1-2 min per kb - Lcp : 1128 bp);
- Third (1X): 72°C for 10min;
- Hold in 4°C.
PCR amplification for difficult amplicons (applied to roxA)
Materials
- 1μl DNA template at about 300 ng μl-1;
- 1.25 μl of each primer forward and reverse (diluted to 20μM) previously designed and purchased;
- 25 μl Q5 High-Fidelity 2X Master Mix (NEB);
- Sterile deionized water quantum sufficit for 50 μl.
Methodology
- In a thermal cycler (BIO-RAD) set the following steps :
First (1X): 98°C for 30s;
Second (30X): 95°C for 10 s; 63°C for 30s (primers Tm calculated by NEB TM calculator - http://tmcalculator.neb.com/#!/); 72°C for 1min (20-30s per kb);
Third (1X): 72°C for 2min;
Hold in 4°C.
- Run a gel electrophoresis to analysis (3 μl) and purification (all remainder reaction)
Methodology
- In a thermal cycler (BIO-RAD) set the following steps :
- Run a gel electrophoresis to analysis (3 μl) and purification (all remainder reaction)
- First (1X): 98°C for 30s;
- Second (30X): 95°C for 10 s; 63°C for 30s (primers Tm calculated by NEB TM calculator - http://tmcalculator.neb.com/#!/); 72°C for 1min (20-30s per kb);
- Third (1X): 72°C for 2min;
- Hold in 4°C.
Gibson assembly
For complete protocol : https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix
Methodology
- ng of insert = kb of insertkb of vector x ng of vector (50 ng usually) x ratio.Ratio for inserts little than 200bp (1:5); for inserts bigger than 200bp use 1:5. Recommended 0.03 - 0.2 pmol; pmol gBlock = weight in ngbase pair x 650 daltons x1000
Pre-inoculum
Methodology
- E. coli cells transformed were plated on LB agar (Sigma) plates supplemented with antibiotic:
chloramphenicol (34 ug/ul) or ampicillin (100ug/ul)
- Grown for 18-20 hours at 37°C.
- Tip 1 colony from the LB agar plates into independent sterile tubes with 5 ml of LB media (1:4) containing antibiotic
- Repeat the previous step twice for 2 different colonies to obtain biological replicate.
- Grown overnight (14 - 16 hours) at 37°C, 80 - 300 rpm.
- chloramphenicol (34 ug/ul) or ampicillin (100ug/ul)
Plate Reader
Optical Density
- Using a clear, flat- or round-bottomed, 96-well plate we measured optical density at 600 nm (OD600).
Methodology
- In each well add LB and pre-inoculum
usually in a proportion 1:10 we obtained OD600 ~ 0.1 with the previous pre-inoculum conditions
- Measure OD600
- If necessary, adjust LB:pre-inoculum proportion to the proper OD600.
- Constitutive promoter: measure OD600
- Inducible promoter: add inducer and measure OD600 in time (8-12 hours)
Fluorescence
- Using a black, flat-bottomed, 96-well plate we measured fluorescence adjusting excitation and emission wavelength according to the fluorescent molecule.
Methodology
- In each well add LB and pre-inoculum
usually in a proportion 1:10 we obtained OD600 ~ 0.1 with the previous pre-inoculum conditions
- Measure OD600
- If necessary, adjust LB:pre-inoculum proportion to the proper OD600.
- Constitutive promoter: measure OD600
- Inducible promoter: add inducer and measure OD600 in time (8-12 hours)
- Induction response must be observed in time due to cellular growth and consequent gene expression. Induction during the mid-log phase of growth might maximize protein expression
- usually in a proportion 1:10 we obtained OD600 ~ 0.1 with the previous pre-inoculum conditions
- usually in a proportion 1:10 we obtained OD600 ~ 0.1 with the previous pre-inoculum conditions
Flow Cytometer
Methodology
- Diluted cultured bacteria to approximately 8 x 106 bacteria mL-1 or OD600 of ~ 0.01 (cell concentration must be in the range of 1x106 to 2 x 107 bacteria mL-1 according to the equipment manufacturer’s recommendation).
- Wash twice with phosphate buffer saline (PBS) at centrifugation 5000 g for 5 minutes.
- Remove the supernatant
- Resuspended the pellet in sheath fluid, same volume as used to obtain OD600 of 0.01.
- Constitutive promoter: measure fluorescence using the appropriate channel
- Inducible promoter: we previously induced the cultured bacteria and used non-induced cultures as control.
Western Blotting
SDS-PAGE running buffer
- 25mM Tris
- 190 mM glycine
- 0.1% SDS
- Check the pH and adjust to pH 8.3
Transfer buffer
- 25mM Tris
- 190mM glycine
- 20% methanol
- Check the pH and adjust to pH 8.3
Ponceau S staining buffer
- 20mM Tris pH 7.5
- 150mM NaCl
Tris-buffered saline Tween 20 (TBST) buffer
- 20mM Tris pH 7.5
- 150mM NaCl
- 0.1% Tween 20
Blocking buffer
- 5% milk in TBST
- Separate the protein sample using electrophoresis gel. Apply the samples with a multicolor broad range marker to ensure the transference.
- Place the gel in 1x transfer for 15 minutos.
- Assemble the transfer sandwich as shown in the image below.
- Place the cassette in a transfer tank and place a ice block in the tank.
- Transfer for 1 hour, 100V, 350mA.
- Stain the blot in ponceau staining buffer to check the transfer quality.
- Rinse of the ponceau with 3 washes with 1x TBST.
- Block with 5% milk in TBST for 1 hour at room temperature.
- Rinse the blot 3 to 5 times with 1x TBST.
- Incubate with the primary antibody solution (anti His-tag) for 3 hours at room temperature.
- Rinse the blot 3 to 5 times with 1x TBST.
- Incubate with the HRP-conjugated secondary antibody for 1 hour at room temperature.
- Rinse the blot 3 to 5 times with 1x TBST.
- Treat the membrane with luminol solution (6ml luminol, 20μl peroxidase).
- Capture the luminescent signals with a CCD camera-based imager.
- Use an image analysis to read the band intensity of the target proteins.