Preparation of chemocompetent cells

Materials

• LB Medium
• CaCl₂ 0.1 M
• CaCl₂ 0.1M + 15% glycerol
• Desired bacterial strain

Steps

1. Inoculate an isolated colony of the desired bacterial strain in a sterile tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
2. Inoculate 100 mL of LB broth with 1 mL of the previous culture (1:100 proportion).
3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
4. Place on ice for 10 minutes.
5. Divide the culture in sterile Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl₂ and incubate on ice for 10 minutes.
7. Centrifuge at 4000 rpm for 10 minutes.
8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl₂ 0.1M + 15% glycerol solution and incubate on ice for 10 minutes.
9. Prepare 200 μl aliquots and store at -80 °C.

Chemocompetent cell transformation

Materials

• Chemocompetent cells
• Plasmid DNA
• LB or SOC medium

Previous Steps

• Prepare a water bath at 42°C.

Steps

1. Thaw a tube of competent cells on ice.
2. Add 5-10 μL of DNA (concentration between 1 pg/mL- 100 ng) to 125μL the competent cells.
3. Incubate the mixture in ice for 30 minutes.
4. Place in the water bath at 42°C for 30 seconds and immediately incubate on ice for 5 minutes.
5. Add 950 μL with SOC or LB medium under sterile conditions.
6. Incubate at 37°C for 60 minutes at 250 rpm.
7. Centrifuge at 5000 rpm for 2 minutes to obtain a pellet.
8. Discard 800 μL from the supernatant and resuspend the pellet in the remaining 200 μL.
9. Spatulate the volume on LB plates with the selection antibiotic and incubate for 16 hours at 37°C.

Annealing of oligonucleotides

Materials

• Oligonucleotides
• Injectable water
• NEBuffer 2.1

Previous Steps

• Prepare a water bath at 95°C.

Steps

1. Perform the corresponding calculations for a desired concentration (μg/μL) and the volume needed to use 5 μg of each oligonucleotide.
2. For each oligonucleotide, make a mix with 1X NEBuffer 2.1 for a total volume of 20 μl with the following materials:
• Injectable water
• NEBuffer 2.1
• Sense oligonucleotide
• Antisense oligonucleotide
3. Incubate the mixture at 95°C for 5 minutes on the water bath.
4. Leave in the water bath to cool down until room temperature is reached.
5. Place on ice until further use.

Plasmid digestion

Materials

• NEBuffer 2.1
• Restriction enzymes
• Injectable water

Steps

1. Make a Master Mix with the following:
• 18 μL of injectable water
• 5 μL of NEBuffer 2.1
• 1 μL of Enzyme 1
• 1 μL of Enzyme 2
2. Add 6 μL of the Master Mix to 6 μL of linearized plasmid in a sterile tube.
3. Incubate at 37° for 2 hours.
4. Inactivate in water bath at 80°C for 20 minutes.
5. Incubate on ice for immediate use or store at -20 oC.

Ligation

Materials

• Injectable water
• T4 DNA ligase buffer (10X)
• T4 DNA ligase
• Aligned oligonucleotides
• Digested linearized plasmid (25ng/μL)

Steps

1. Prepare a master mix (if necessary) with the following components:
• Digested plasmid
• Water °MB
• T4 DNA ligase
• T4 DNA ligase buffer
2. Distribute the master mix among the reaction tubes and place the oligonucleotides aligned to have a final volume of 10 μL per reaction.
3. Leave incubating at room temperature overnight and transform the next day.

Lowry Method for protein quantification

Standard curve protocol

1. To identify the protein concentration, quantify a BSA standard curve.
2. Preparation of samples with known proteins for a BSA stock (1mg/ml):

Table 1. Known protein concentrations with the volumes of each reactant for its preparation.

Sample	BSA (μg)	BSA vol (1ug/ul)	H2O (ul)	C Reactant (ul)	Folin Reactant (ul)
1	0	0	100	500	50
2	10	5	95	500	50
3	20	10	90	500	50
4	30	15	85	500	50
5	40	20	80	500	50
6	50	25	75	500	50
7	60	30	70	500	50
8	70	35	65	500	50
9	80	40	60	500	50
10	110	55	45	500	50
11	120	60	40	500	50

3. After the samples of each concentration are prepared, add 500 μl of solution “C” (Biuret reactant) to each one and incubate for 10 minutes at room temperature.
4. Add 50 μl of Folin reactant to each sample and then incubate 45 minutes at room temperature.
5. Add 150μl per sample in a 96-well plate and read absorbance.

Protein quantification for the curve sample protocol

1. Resuspend samples in lysis buffer RIPA (200μl).
2. Dilute 1:20 using 10μl of sample in 190μl of milliQ water.
3. Add 1ml of “solution C” to the diluted samples and incubate for 10 minutes at room temperature.
4. Add 10μl of Folin reactant per sample and resuspend. Incubate 45 min at room temperature.
5. In a 96 well plate add 150μl per sample and read absorbance at 660.

Plasmid extraction

Materials

• Transformed E. coli DH5a cells.
• LB + CAM
• STET Buffer
• Lysozyme
• RNAse A
• 3 M Sodium acetate
• Isopropanol
• 70% ethanol
• Injectable water

Steps

1. Seed an isolated colony of each transformed Escherichia coli strain in 10 mL of LB+CAM in a test tube and incubate for 16 hours at 37oC at 260 rpm.
2. Centrifuge all the culture in 2 mL sterile microtubes at 13,000 rpm for 1 minute in order to retrieve the pellet.
3. Remove the supernatant and wash the pellets without resuspending with 350μL of STET Buffer (0.1M NaCl, 10 mM TRIS-HCl pH 8, 1 mM EDTA pH 8 and Triton X-100 5%)
4. Resuspend with 350 μL of STET Buffer and add 25μL of fresh lysozyme (2 mg/mL).
5. Incubate for 4 minutes at room temperature, and then place in a water bath at 100oC for 1 minute.
6. Centrifuge at 12,000 rpm for 10 minutes and remove the pellet with a stick.
7. Add 5 μL of RNAse A and incubate for 20 minutes at 37°C .
8. Add 75 μL of sodium acetate 3M pH 5.2 and 250 μL of isopropanol and incubate for 10 minutes at room temperature.
9. Centrifuge at 12,400 rpm for 10 minutes and discard the supernatant.
10. Wash the pellet twice with 750 μL of 70% ethanol to eliminate salts.
11. Resuspend the pellet in 150 μL of water for injections.

SDS-PAGE

Steps

1. Resuspend each sample in Laemmli buffer (B-Mercaptoethanol, 0.1%, Bromophenol blue 0.0005% ,Glycerol, 10%, SDS (electrophoresis-grade) 2% and Tris-HCl, 63 mM (pH 6.8). Make the necessary calculations so all samples have the same amount of protein.
2. Homogenize samples with the polytron.
3. Place the samples on a water bath at 100oC for 5 minutes.

Pouring the resolving gel

1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and spacers.
2. Once the gel holder has been assembled, it is verified that there is no leak, the boundary of the separating phase is then marked 0.5 cm below the teeth of the comb.
3. Prepare 10 mL of separating phase. Due to the size of RFP (26 kDa), 10% or 12% gel would do.

Table 1: Reagents required to prepare the separating phase in different % gels.

Acylamide percentage	6%	8%	10%	12%	15%

4. Add 5 mL the separating phase mixture taking care not to exceed the previously marked limit.
5. So that the phase is even and without bubbles, add a layer of ethanol, which is not miscible with the above mixture.
6. When the separating phase has gelled, the ethanol is removed with the help of a filter paper.
7. Prepare 5 mL of the concentrating phase.

Table 2: Reagents required to prepare the concentrating phase.

H2O	2.975ml

8. The concentrating phase is added and the comb is placed so that there are no empty spaces. After waiting until it gels, the comb is removed.
9. Place the gel in the electrophoresis, cover with the running buffer.
10. Load the samples and the molecular weight in the wells.
11. Run the gel at given conditions.
12. Stain with Coomassie Blue dye overnight.
13. Destain with Destaining Solution (30% Methanol, 7% acetic acid) for 30 minutes. After the time is up, replace the solution with fresh one. Repeat until the gel is visible.

Plasmid extraction with Monarch ® Plasmid DNA Miniprep Kit from New England Biolabs TM

Previous step

• Prepare Plasmid Wash Buffer 2, add 4 volumes of ethanol (> 95%) to one volume of Buffer. Add 24 mL of ethanol for a final volume of (30 mL) of Plasmid Wash Buffer 2.

*All centrifugations steps should be carried out at 13,000 rpm.

Steps

1. Pellet 10 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 400 μL plasmid Resuspension Buffer (B1). Vortex to ensure cells are completely resuspended. There should be no visible clumps.
3. Add 400μl Plasmid Lysis Buffer (B2), gently invert tube 5-6 times, and incubate at room temperature for 1 minute. Color should change to dark pink, and solution will become transparent and viscous. Do NOT vortex.
4. Add 800μl of Plasmid Neutralization Buffer (B3), gently invert tube until neutralized, and incubate at room temperature for 2 minutes. Sample is neutralized when color is uniformly yellow and precipitate forms. Do NOT vortex.
5. Centrifuge lysate for 10 minutes.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5ml microfuge tube. Use care to ensure that the tip of the column does NOT come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min.
10. Add 100 μl of injectable water to the center of the matrix. Wait for 1 minute, then spin for 1 min to elute DNA. A second eluted DNA was realized with 50 μl of water.

DNA band purification with GenElute TM Gel Extraction Kit from Sigma-Aldrich.

Materials

• Scalpel
• Ethanol 100%
• Isopropanol 100%
• 3M Sodium Acetate Buffer, pH 5.2
• Water bath at 60°C
• Molecular Biology water
• All the centrifuge are performed at 16,000 xg or 13,155 rpm

Previous steps

• Prepare the Wash Solution Concentrate G with 48 mL of EtOH 100%

Steps

1. Excise the DNA fragment of interest from the agarose gel with the scalpel.
2. Weigh the gel slice in a microtube.
3. For every 100 mg of agarose gel, add 300 mL of Gel Solubilization Solution.
4. Incubate the gel mixture at 50-60 °C for 10 minutes, or until the agarose has dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel.
5. Prepare the binding column in a microtube of 2 mL, add 500 μL of the Column Preparation Solution, centrifuge for 1 min and discard flow-through liquid.
6. If the mixture is red, add 10 μL of Sodium Acetate Buffer, until the mixture turns yellow.
7. Add 1 gel volume isopropanol and mix.
8. Load the gel solution to the binding column. If the volume of the gel mixture is >700 mL, load the sample onto the column in 700 mL portions. Centrifuge for 1 minute after loading the column each time. Discard the flow-through liquid.
9. Add 700 μL of Wash Solution to the binding column, centrifuge 1 minute and discard the flow- through. Centrifuge again 1 minute.
10. Elute DNA, transfer the column to a new microtube, add 50 μL of Elute Solution, incubate for 1 minute and centrifuge 1 minute.

Polymerase Chain Reaction

Steps

1. Prepare the Master Mix for the PCR reaction adding
• 0.75μL 50 mM MgCl2
• 22. 5 μL PCR Buffer 10X
• 0.5 μL 10 mM dNTP Mix
• 1 μL 10 mM FWD Primer
• 1 μL 10 mM REV Primer
• 0.2 μL Taq DNA Polymerase
• 18.05 μL MBo Water
2. Add the Master Mix and 1 μL of DNA template for each reaction. Final volume: 45 μL per reaction.
3. Reactions are placed in the thermal cycler with the following program:
• 94°C for 5 minutes
• 31 cycles:
• 94°C for 30 seconds
• 55°C for 30 seconds
• 72°C for 30 seconds
• 72°C for 5 minutes
• 12°C indefinitely
4. For PCR product analysis, an agarose gel electrophoresis must be performed.

Note: Number of cycles and alignment temperatures may vary in each case.

Preparation of chemocompetent cells

Materials

• LB Medium
• 01. M CaCI2
• CaCL2 0.1M + 15% glycerol
• Sterile Falcon tubes

Steps

1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
4. Place on ice for 10 minutes.
5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
7. Centrifuge at 4000 rpm for 10 minutes.
8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
9. Prepare 200 μl aliquots and store at -80 °C.