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.

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.

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.

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.

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.

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.

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.