Team:Brasil-USP/Notebook/protocols

Protocols

Notebook

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
      •    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.
  • Resuspension
      •    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.
  • Lysis
      •    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.
  • Neutralization
      •    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.
  • Washing
      •    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.
  • Elution of plasmidial DNA
      •    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

Ligation reaction (Cohesive ends)



Reference: 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.

    • Methodology


    • 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;
  • Heat shock in E. coli DH5α with 10 μl of the reaction.

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



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 following 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.

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).

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)

Gibson assembly



For complete protocol : https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix


Methodology



  • Reaction

      • \[ ng_{insert} =\left (\frac{kb_{insert}}{kb_{vector}} \right )\left (ng_{vector} \right )\left (ratio \right )\] Ratio for inserts smaller than 200bp (1:5); for inserts bigger than 200bp use 1:5. Recommended 0.03 - 0.2 pmol; pmol \[ gBlock =\left (\frac{weight}{bp x 650 Daltons} \right )\left (1000 \right )\] use weight in ng
  • Incubate at 50°C for 15 minutes;
  • Heat shock in E. coli DH5α with 10 μl of the reaction.

    • 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.

    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

    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.

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