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

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 Doc^TM 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).

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).

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.

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

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.