Difference between revisions of "Team:Brasil-USP/Notebook/protocols"
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<p>For complete protocol : https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix</p> | <p>For complete protocol : https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix</p> | ||
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<h2>Methodology</h2> | <h2>Methodology</h2> | ||
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− | + | <ul>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</ul> | |
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Revision as of 01:43, 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.
- ransfer 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