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Protocols

Notebook

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
      • 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
      • 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.
  • 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)



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.

    • Methodology


    • Set up the following reaction in a microcentrifuge tube on ice:


        • * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
        q.s. = quantum sufficit
        ** ng of insert = [tex]\frac{kb of insertkb}{kb of vector}[/tex]x ng of vector (50 ng usually) x ratio. The ratio was considered to be 3, but it can vary according to the vector
    • For cohesive ends, incubate at 22°C for 3 hours + 16°C for 9 hours.

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).

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