Sonication protocol

1. To each sample add 5 mL of 50 mM tris
2. Sonicate at 70 % repeating 3 cycles of 15 seconds on, 60 seconds off
3. Centrifuge at 9,838 g during 10 min at 4 ºC

12% resolving SDS-PAGE gel

1. To a falcon tube add the components shown in the table in order
2. Using a micropipette add the prepared solution until below the top of the gel mold
3. Add ethanol until fully covered

Reagent	Quantity [μL]
Acrylamide 30%	3, 250
Tris pH 8.8	1, 005
Distilled water	3, 370
SDS 10%	78
APS	45
TEMED	4

10% stacking SDS-PAGE gel

1. To a falcon tube add the components shown in the table below
2. If resolving gel is solidified, remove ethanol by tipping
3. Using a micropipette add the prepared solution until the top of the gel mold
4. Add the comb ensuring a good fit

Reagent	Quantity [μL]
Acrylamide 30%	500
Tris pH 6.8	200
Distilled water	224
SDS 10%	30
APS	11.5
TEMED	2.25

Notes

• Add Ammonium persulfate (APS) and Tetramethylethylenediamine (TEMED) right before adding gel to the mold as it will begin polymerizing
• Use the remaining mixture in the tube to determine when gel has solidified

Miniprep with QIAprep Spin Miniprep Kit

1. Pellet overnight cultures by centrifugation (13,000 rpm, 2 min) adding 1.5 mL at a time
2. Decant supernatant
3. Add 250 μL of Cell Lysis Solution and invert until clear
4. Add 10 μL of Alkaline Protease Solution and invert to mix
5. Incubate at room temperature for 5 min
6. Add 350 μL of Neutralization Solution and invert until homogenized
7. Centrifuge at maximum speed (11.4 g) for 10 min
8. Assemble spin column and add lysate
9. Centrifuge at maximum speed for 1 min
10. Discard flow through
11. Add 750 μL of Wash Solution
12. Centrifuge at 11.4g for 1 min
13. Add 250 μL of Wash Solution
14. Centrifuge at maximum speed for 2 min
15. Transfer spin column to a new and sterile 1.5 mL Eppendorf tubes
16. Add 80 μL of nuclease free water
17. Centrifuge at maximum speed for 1 min
18. Discard column
19. Measure yields on Nanodrop
20. Store at -20 ºC until further usev

Note

• When loading the blank on the Nanodrop for calibration use the same liquid (buffer or water) used to elute

Cleared lysate

1. Harvest cells by centrifugation at 4,000 rpm, 10 min, 4 ºC
2. Decant supernatant
3. Resuspend in 250 μL of Cell Lysis Solution
4. Invert 4 times to mix
5. Add 10 μL of Alkaline Solution
6. Invert 4 times to mix
7. Incubate at room temperature for 5 min
8. Add 350 μL of Neutralization Solution
9. Invert 4 times to mix
10. Centrifuge at 14.1 g (top speed) for 10 min

DNA Plasmid binding

1. Assemble spin column and collection tube
2. Add supernatant into the column
3. Centrifuge at top speed for 1 min
4. Discard flow through

Washing

1. Add 750 μL of Wash Solution
2. Centrifuge at top speed for 1 min
3. Discard flow through
4. Add 250 μL of Wash Solution
5. Centrifuge at top speed for 2 min
6. Discard flowthrough
7. Centrifuge at top speed for 2 min
8. Discard flowthrough

Elution

1. Transfer spin column to a sterile 1.5 mL eppendorf
2. Add 100 μL room-temperature nuclease free water
3. Centrifuge at top speed for 1 min
4. Discard column
5. Measure yields on Nanodrop

Miniprep – Fast-n-easy plasmid miniprep kit PP-2045

1. Harvest cells by centrifugation (4000 rpm, 10 min, 4 ºC)
2. Add 300 μL of Lysis Buffer and resuspend with micropipette
3. Add 300 μL of Neutralization Buffer
4. Invert 5 times to mix
5. Centrifuge (10,000g, 5 min)
6. Assemble binding column
7. Add 100 μL of Activation Buffer into the column
8. Centrifuge column (10,000 g, 30 sec)
9. Micropipette supernatant to activated column
10. Centrifuge (10,000 g, 30 sec)
11. Discard flowthrough
12. Add 500 μL of Washing Buffer
13. Centrifuge (10,000g, 30 sec)
14. Discard flowthrough
15. Centrifuge (10,000 g, 30 sec)
16. Discard flowthrough
17. Place column into clean 1.5 mL tube
18. Add 100 μL of Nuclease Free H₂O at 60 ºC
19. Incubate 5 min at 60 ºC
20. Centrifuge (11, 000 rpm, 1 min)
21. Quantify yields on Nanodrop

Notes

• Some deviations from suppliers instructions due to advice from PI
• Secondary wash with 700 μL of Washing Buffer is not done
• During elution step: instead of 30 - 50 μL of Elution Buffer, 100 μL of warm nuclease-free H₂O were added
• During elution step: prior to final centrifugation, a 5 minute incubation at 60 ºC was added
• When loading the blank on the Nanodrop for calibration use the same liquid (buffer or water) used to elute

Ligation

1. Add the reagents of the table in order

Reagent	Quantity [μL]
Insert	Dependent on ratio
T4 DNA ligase buffer	2
Vector	Dependent on ratio
T4 DNA ligase	1
H2O	To 20 μL

2. Incubate at room temperature for 1 hour
3. Proceed to transformation

Notes

• Any remaining ligation that wasn't used for the transformation was left overnight at room temperature then stored at 4 ºC
• NEB calculator was used to determine possible ratios

Preparing LB + carbenicillin plates

1. Add 0.5 g of agarose to 25 mL of distilled water (20 g L^-1)
2. Homogenize by shaking
3. Sterilize at 121 °C
4. Once cool to touch add 25 μL of carbenicillin (1 μL mL^-1)
5. Pour into the plate and let harden

Note

• Multiply agarose, distilled water, and carbenicillin based on number of plates to prepare

QIAprep Spin Miniprep Kit

1. Cut target band as accurately as possible
2. Add QG buffer at a 1:3 ratio
3. Melt the gel completely in a 50 ºC water bath
1. Heat up nuclease free water simultaneously
2. Invert every 2 minutes
4. Add 1 gel volume of isopropanol
5. Assemble column
6. Add 500 μL of sample to the column
7. Centrifuge for 1 min at 13, 500 rpm
8. Discardo flowthrough
9. Repeat steps 6 - 8 until all sample is run through the column
10. Add 750 μL of PE buffer
11. Centrifuge for 1 min at 13, 500 rpm
12. Discard flowthrough
13. Incubate at room temperature for 2 min
14. Centrifuge for 1 min at 13, 500 rpm
15. Transfer column to clean 1.5 mL eppendorf
16. Add 50 μL EB buffer
17. Centrifuge for 1 min at 13, 500 rpm
18. Measure yield on Nanodrop

Note

• When loading the blank on the Nanodrop for calibration use the same liquid (buffer or water) used to elute

Coomassie staining SDS-PAGE

1. Carefully remove gel from glass molds and place gel in plastic container
2. Add coomassie blue stain to gel until fully submerged
3. Let stain for at least 1 hour at gentle agitation (40 rpm)
4. Remove coomassie blue and submerge in distilled water with gentle agitation overnight

Note

• When adding coomassie stain avoid pouring directly on the gel to prevent uneven staining

Colony PCR

1. Add 50 μL of LB to a microtube
2. In a separate microtube add the reagents listed in the following table

Reagent	Amount [μL]
Q5 master mix	25
DNA	< 1 000 ng
FW primer	5
RV primer	5
nuclease free H2O	to 50

3. Pick a colony with a sterile micropipette tip
4. Leave the tip in the PCR tube for 15 seconds
5. Transfer the tip to the LB tube and leave for 30 seconds
6. Put PCR tube in thermocycler with the following conditions (35 cycles)

Temperature [ºC]	Duration
98	00:00:30
98	00:00:10
61	00:00:30
72	00:01:15
72	00:02:00
4	∞

Calcium-competent cells

1. Incubate 50 mL of E. coli on ice for 1 hr
2. Harvest cells by centrifuging (4,000 rpm, 10 min, 4 ºC)
3. Decant supernatant and resuspend in 10 mL of cold CaCl₂
4. Centrifuge 4,000 rpm, 10 min, 4 ºC
5. Decant supernatant and resuspend in 1 mL cold 0.1 M CaCl₂ + 15 % glycerol
6. Make 50 μL aliquots and store at -80 ºC

Acetone precipitation for SDS-PAGE (sample preparation)

1. To 100 μL of sonication supernatant add 700 μL of cold acetone
2. Incubate at -20 ºC for at least 2 hours
3. Briefly spin down samples
4. Remove excess acetone with syringe and discard
5. Evaporate remaining acetone at 30 ºC for 20 minutes
6. Resuspend in 100 μL tris buffer
7. To each sample 50 μL of Laemmli and homogenize by pipetting
8. Add samples to 96 ºC water bath for 5 min
9. Transfer to ice for 5 min
10. Load samples onto gel

8% resolving SDS-PAGE gel

1. To a falcon tube add the components shown in the table in order
2. Using a micropipette add the prepared solution until below the top of the gel mold
3. Add ethanol until fully covered

Reagent	Quantity [μL]
Acrylamide 30%	1, 200
Tris pH 8.8	1, 950
Distilled water	2, 700
SDS 10%	150
APS	15
TEMED	6

4% stacking SDS-PAGE gel

1. To a falcon tube add the components shown in the table below
2. If resolving gel is solidified, remove ethanol by tipping
3. Using a micropipette add the prepared solution until the top of the gel mold
4. Add the comb ensuring a good fit

Reagent	Quantity [μL]
Acrylamide 30%	375
Tris pH 6.8	375
Distilled water	2, 925
SDS 10%	7.5
APS	12
TEMED	6

Notes

• Add APS and TEMED right before adding gel to the mold as it will begin polymerizing
• Both layers of the gel will take longer to solidify compared to a 12% and 10% gel

8% resolving SDS-PAGE gel

1. After assembling the gel mold, add the following reagents to a 15 mL Falcon tube

Reagent	Quantity [μL]
Acrylamide 30%	1, 200
Tris pH 8.8	1, 950
Distilled water	1, 179
SDS 10%	150
APS	15
TEMED	6

2. Invert several times to homogenize and then add it to the mold using a 1000 μL micropipette (up to the first line that forms)
3. Add ethanol up to the edge of the glass

4% stacking SDS-PAGE gel

1. Add the following reagents to a 15 mL Falcon tube

Reagent	Quantity [μL]
Acrylamide 30%	375
Tris pH 6.8	375
Distilled water	1, 844
SDS 10%	150
APS	24
TEMED	12

2. After confirming that the resolving gel has polymerized (approximately 30 minutes), remove the ethanol by inverting the mold
3. Add it using a 1000 μL micropipette into the mold until it reaches the edge of the glass
4. Place the comb inside the mold and wait for the gel to polymerize (approximately 30 minutes)
5. When the gel has polymerized, place it inside the chamber, add running buffer, and remove the comb

Notes

• Add APS and TEMED right before adding gel to the mold as it will begin polymerizing
• Both layers of the gel will take longer to solidify compared to a 12% and 10% gel
• On several occasions, the gel was prepared with an acrylamide concentration of 5.97% for the resolving gel and 2.98% for the stacking gel. To achieve this, the following quantities were added to the Falcon tubes

Reagent	Quantity [μL]
Acrylamide 30%	1, 200
Tris pH 6.8	1, 950
Distilled water	2, 700
SDS 10%	150
APS	15
TEMED	6

Reagent	Quantity [μL]
Acrylamide 30%	275
Tris pH 8.8	375
Distilled water	2, 925
SDS 10%	75
APS	12
TEMED	6

Sample preparation

1. To 100 μL of sonication supernatant or samples from protein purification, add 700 μL of cold acetone
2. Incubate at -20 ºC for at least 2 hours
3. Briefly spin down samples until reaching maximum speed in the microcentrifuge (13,500 rpm)
4. Remove excess acetone with a syringe and discard
5. Evaporate remaining acetone at 30 ºC for 20 minutes
6. Resuspend in 100 μL TRIS buffer 10 mM
7. To each sample, add 50 μL of Laemmli and homogenize by pipetting
8. Incubate for 5 min at room temperature
9. Add samples to 96 ºC water bath for 5 min
10. Load samples onto gel

Resin preparation

1. Add 10 mL of buffer A (100 mM TRIS, 300 mM NaCl, 2 mM Imidazole) and 0.5 mL resin to a 15 mL Falcon tube
2. Shake for 5 minutes
3. Centrifuge for 5 minutes at 500 rpm, 4°C
4. Discard the supernatant and retain the resin

AtPCS purification

1. Add the supernatant recovered from sonication to the tube with the resin
2. Resuspend the precipitates with 4 mL of distilled water, take a sample (500 μL), and transfer it to a microcentrifuge tube (precipitate)
3. Shake for 30 minutes at 80 rpm
4. Centrifuge for 5 minutes at 500 rpm, 4°C
5. Take a sample (500 μL) of the supernatant and transfer it to a microcentrifuge tube (flow through)
6. Discard the remaining supernatant using a suction device
7. Add 10 mL of buffer B (100 mM TRIS, 300 mM NaCl, 10 mM Imidazole)
8. Invert for 5 minutes to homogenize
9. Centrifuge for 5 minutes at 500 rpm, 4°C
10. Take a sample (500 μL) of the supernatant and transfer it to a microcentrifuge tube (first wash)
11. Discard the rest of the supernatant using a suction device
12. Add 10 mL of buffer B (100 mM TRIS, 300 mM NaCl, 10 mM Imidazole)
13. Invert for 5 minutes to homogenize
14. Transfer the tube contents to a 10 mL syringe with a filter paper inside
15. Take a sample (500 μL) of the filtrate and transfer it to a microcentrifuge tube (second wash)
16. Add 1 mL of buffer E (100 mM TRIS, 300 mM NaCl, 200 mM Imidazole) and recover it in a microcentrifuge tube (eluted protein)
17. Recover the resin from the syringe by adding distilled water and transfer it to the 15 mL tube
18. Store samples and resin at 4°C

Protocol:

1. Overnight culture and pre-culture
1. Pour 3 ml of LB medium into a sterile 15 ml conical tube and inoculate with 1 Escherichia coli colony or 50 μL of liquid inoculum. Incubate at 37°C and 200 rpm overnight
2. Add 0.5 ml of the overnight culture into 49.5 of sterile LB medium, in a sterile Erlenmeyer flask
3. Incubate at 37°C with 200 rpm during 2~4h, until the optical density of the culture reading at 600 nm (OD₆₀₀) reaches 0.3~0.4. Once the OD₆₀₀ reaches 0.15, it is recommended to take measurements each 30 min; and if it is over 0.21, each 15 min
2. CaCl₂ washes
1. Keep all reagents at 4°C, on ice
2. Transfer the E. coli culture into a sterile 50 ml conical tube. Incubate on ice for 1 h
3. Centrifuge at 4°C with 4,000 rpm for 10 min, discard the supernatant
4. Resuspend the cell pellet in 5 ml of cold 0.1 M CaCl₂ and incubate on ice for 45 min
5. Centrifuge at 4°C with 4,000 rpm for 10 min, discard the supernatant
6. Resuspend the cell pellet in 600 μL of 0.1 M CaCl₂ with 15% glycerol*
*To prepare 0.6 ml of 0.1 M CaCl₂ + 15% glycerol, mix 112.5 μL of 80% glycerol with 487.5 μL of 0.1 M CaCl₂
7. Prepare ten 60 μL aliquots of competent cells in sterile 1.5 ml or 1.0 ml microtubes and refrigerate at -80°C until use

Protocol

1. Place all reaction components in a 0.2 ml microtube and quickly transfer the reaction to the thermocycler, according to the table below (for smaller volumes, adjust the volume of the Master Mix and primers proportionately). All components should be mixed prior to use

Component	Amount [μL]
Q5® High-Fidelity 2X Master Mix	25
Forward primer	2.5
Reverse primer	2.5
Template DNA	Variable (20-100 ng)
Nuclease-free H2O	To 50 μL
Total volume	50

2. onfigure the thermocycler according to the length of the fragment to amplify and to the annealing temperature of the primers (https://tmcalculator.neb.com/#!/main). The usual configuration for the purpose of amplifying the AtPCS gene is shown as follows

Stage		Temperature	Time
Initial denaturation		98°C	30 s
Denaturation	[35 x]	98°C	10 s
Annealing		61°C*	30 s
Extension		72°C	1.5 min**
Final Extension		72°C	2 min
Hold		4°C	∞

* According to annealing temperature of the primers in use
** 30 s/kb, then multiplied by 2
3. After the reaction is done, perform agarose gel electrophoresis to verify the integrity and identity of the amplicon. Further purification can be performed from the gel or directly from the reaction mixture

Current protocol

1. Harvest cells from overnight culture (1-10 mL) by centrifugation at 4,000 rpm, 10 min, 4 ºC
2. Discard supernatant
3. Resuspend in 250 μL of Cell Resuspension Solution
4. Place the resuspended cells in a microcentrifuge tube
5. Add 250 μL of Cell Lysis Solution and invert 4 times to mix
6. Add 10 μL of Alkaline Protease Solution and invert 4 times to mix
7. Incubate at room temperature for 5 min
8. Add 350 μL of Neutralization Solution and invert 4 times to mix
9. Centrifuge at top speed (13 500 rpm) for 10 min
10. Assemble spin column and collection tube
11. Add 700 - 800 μL of cleared lysate into the spin column
12. Centrifuge at top speed (13 500 rpm) for 1 min and discard flowthrough
13. Add 750 μL of Wash Solution with EtOH added
14. Centrifuge at top speed (13 500 rpm) for 1 min and discard flowthrough
15. Repeat steps 13 and 14 with 250 μL of Wash Solution with EtOH added
16. Centrifuge at top speed (13 500 rpm) for 2 min and discard flowthrough
17. Transfer spin column to a sterile 1.5 mL microtube
18. Add 80 μL of nuclease free water at 60 °C to the spin column
19. Centrifuge at top speed (13 500 rpm) for 1 min
20. Discard column
21. Measure yields on Nanodrop

Current protocol

1. Harvest cells from overnight culture (1-10 mL) by centrifugation at 4,000 rpm, 10 min, 4 ºC
2. Discard supernatant
3. Resuspend pellet in 300 μL of Lysis Buffer by vortex (around 1 min)
4. Transfer the sample to a new microcentrifuge tube
5. Add 300 μL of Neutralization Buffer and invert 4-6 times
6. Centrifuge for 5 min at 10,000 g (the color of the sample should change to yellow)
7. Place a column into a 2 mL collection tube
8. Add 100 μL of Activation Buffer to the column
9. Centrifuge for 30 sec at 10,000 g
10. Place the supernatant from step number 6 into the previously activated column
11. Centrifuge for 30 sec at 10,000 g and discard the supernatant
12. Add 500 μL of Wash Buffer (with EtOH) to the column
13. Centrifuge for 30 sec at 10,000 g and discard the supernatant
14. Centrifuge for 2 min at 10,000 g to remove residual buffer
15. Place the column inside a microcentrifuge tube
16. Add 100 μL of nucleases-free water to the center of the column
17. Incubate for 5 min at 60 ºC
18. Centrifuge for 1 min at 11,000 g
19. Quantify concentration using Nanodrop

Name of the experiment
Gel extraction protocol: QIAquick Gel Extraction Kit

Brief description of the procedure
The QIAquick Gel Extraction Kit procedure involves running an agarose gel, cutting out the desired DNA fragment, dissolving the gel piece, binding DNA to a QIAquick silica membrane, washing, and eluting the DNA; resulting in a purified DNA sample

Current protocol

1. Cut the band(s) and place it/them into a microcentrifuge tube
2. Weigh the tube with the band and verify that the weight is within the range of 100-200 mg approximately after subtracting the weight of the empty tube (approximately 1 g)
3. Add 700 μL of buffer QG to the tube with the band
4. Incubate at 50°C for 10 minutes. It is recommended to vortex the tube every 3 minutes during incubation and at the end of the incubation
5. After ensuring that the sample is homogeneous, transfer it to a kit column. (It may be necessary to divide the volume of the tube to transfer it entirely to the column, so proceed with step 6 and repeat the process with the rest of the sample)
6. Centrifuge for 1 minute at maximum speed (13500 rpm)
7. Discard the supernatant and repeat with the remaining samples
8. Add 750 μL of buffer PE containing ethanol to each column
9. Centrifuge for 1 minute at maximum speed
10. Discard the supernatant
11. Centrifuge for 2 minutes at maximum speed
12. Transfer the column to a new microcentrifuge tube
13. Elute with 40-50 μL of nuclease-free warm water. It is important to make sure to add the water directly into the center of the column
14. Incubate for 5 minutes at 60°C
15. Centrifuge for 1 minute at maximum speed
16. Quantify using Nanodrop

Notes

• The protocol provided by QIAGEN for using the kit indicates that for each gel volume (exemplified as 100 mg), 3 volumes of QG buffer are added. As previously stated in the protocol, the final amount was standardized, and 700 LL of buffer are used for samples with an approximate weight of 100-200 mg
• The instruction to vortex during the first incubation is to ensure that no gelified remains of the sample are left before transferring it to the column
• It is essential to mention that after adding the QG buffer and starting the first incubation, preferably no more than 20 minutes should pass before centrifuging on the column for subsequent washing. This should be considered when working with multiple samples
• In the current protocol, the use of isopropanol after the first incubation was omitted, as similar results have been obtained with or without it. This step is optional
• The current protocol includes an additional centrifugation step (Step 11) after adding the PE buffer. This is done to minimize any remaining traces of PE buffer in the column
• Finally, a 5-minute incubation at 60°C was added after elution, which has resulted in improved DNA quantification results

Protocol

1. For a 50 μL reaction, add the following components into a 0.2 ml microtube:

Component	Amount [μL]
DNA	Variable (~1 µg)
10X NEBuffer	5.0
Enzyme 1	1.0
Enzyme 2	1.0
Nuclease-free H2O	To 50 μL
Total volume	50

2. Incubate overnight at 37°C
3. Inactivate the enzymes at 65°C for 20 min
4. Perform agarose gel electrophoresis of the sample to identify the relevant DNA fragment and purify it

Protocol

1. Define the amount of vector to use and the vector-insert proportion
2. Place the reagents into a 0.2 ml microtube according to the following table:

Component	Amount [μL]
Vector	Variable (Ideally 100 ng)
Insert	Variable
T4 DNA ligase 10X buffer	2.0
T4 DNA ligase	1.0
Nuclease-free H2O	To 20 μL
Total volume	20

3. Leave the reaction at room temperature overnight
4. After 1 hour, take 5-10 μL of the reaction and transform competent cells or store at -20°C
5. The next day, take another 5-10 μL sample and transform competent cells or store at -20°C

Colony PCR

1. At sterile conditions, add 20 µL of LB to a PCR microtube (repeat for the total number of selected colonies for amplification)
2. Prepare a master mix depending on the samples (considering a final reaction volume of 15 µL):

Reagent	Amount per sample [μL]
Q5 master mix	7.7
FW primer	0.75
RV primer	0.75
nuclease free H2O	to 15

3. Distribute the master mix into PCR microtubes (one for each colony plus a positive control)
4. Using a sterile toothpick or micropipette tip, pick up the first colony and place it into the first microtube with the master mix
5. Rotate the toothpick or tip inside the tube about twenty times and then transfer it to the microtube with LB medium
6. Repeat the last two steps with the rest of the selected colonies
7. For the positive control, take 1 µL of the sample and place it in a microtube with the master mix
8. Remove the toothpicks or tips from the microtubes with LB medium and store them at 4°C
9. Place the PCR tube in the thermocycler with the appropriate conditions depending on the length of the gene to be amplified and the Tm of the primers (35 cycles):

Temperature [ºC]	Duration
98	00:00:30
98	00:00:05 - 00:00:10
50-72	00:00:10 - 00:00:30
72	00:00:20 - 00:00:30
72	00:02:00
4	∞

Preculture

1. At sterile conditions, add 5 mL of LB medium and 5 μL of kanamycin antibiotic to a 50 mL Falcon tube
2. Select a colony of Escherichia coli BL21 (containing the plasmid with the gene) and pick it up with a metal loop
3. Submerge the tip of the loop into the tube with medium and antibiotic, and shake until no traces of the colony are visible
4. Repeat the steps above with the remaining chosen colonies
5. Place the tubes in an incubator at 37°C, 200 rpm, overnight

Inoculation

1. At sterile conditions, place 100 mL of LB medium in a sterile flask (if purification will be done after lysis) or 20 mL in a 50 mL Falcon tube (if no purification is planned)
2. Inoculate with 1 mL or 200 μL of the preculture
3. Incubate at 37°C, 200 rpm until the optical density reaches 0.5-0.6
4. Store at 4°C until the induction moment

Induction with IPTG

1. At sterile conditions, add 40 μL or 8 μL to achieve a concentration of 0.4 mM IPTG
2. Incubate tubes at 25°C (sometimes also done at 16°C), 200 rpm, overnight

Cell harvesting

1. Centrifuge the tubes at 4000 rpm, 10 minutes, 4°C
2. Discard the supernatant
3. Resuspend the pellets in 5 mL of buffer A (100 mM TRIS, 300 mM NaCl, 2 mM Imidazole)

Cell lysis by sonication

1. Sonicate at 75%, repeating 3 cycles of 15 seconds on, 60 seconds off. In some cases, 5 cycles of 30 seconds were also used
2. Centrifuge at 4000 rpm, 10 minutes, 4°C
3. Store the supernatants at 4°C and the pellets at -20°C

Protocols

1. Weigh 5 g of agarose on a weighing tray
2. Measure 50 ml of 1X TAE buffer on a graduated cylinder
3. Pour the buffer into an Erlenmeyer flask and add the agarose. Microwave in short intervals (i.e. less than 1 min) until the agarose dissolves. The solution should be clear, with no agarose granules left
4. Quickly, pour the liquid into a gel mold tray with a comb. Wait 25-30 min for the gel to set
5. Remove the comb carefully and transfer the gel into the electrophoresis tank. The comb side should be next to the negative electrode
6. Fill the tank with 1X TAE buffer up to the maximum volume line. The gel is now ready for the samples to be loaded
7. To load the sample, mix the desired amount of sample with the corresponding amount of 6X loading dye. Place the samples carefully into the wells. It is important to note that SYBR has been added to the dye to achieve fluorescence of DNA present in the samples; thus, from the moment the dye is used until the gel is done running, the gel must be protected from light
8. Close the tank, making sure that the black electrode is on the side of the samples and the red electrode is on the opposite side. Verify that the electrodes are plugged in correctly into the power supply
9. Program the power supply as needed. In our lab, we use 60 V for 15 min and then 80 V for 55 min. Press start
10. Once the time is over, turn off the power supply, remove the lid of the tank and transfer the gel onto an UV transilluminator
11. Turn the transilluminator on to visualize the DNA samples on the gel
12. Recover the relevant samples or discard the gel. The gel can be reused once or twice for purposes such as visualizing PCR products or digestion reactions, but not if further purification is needed

Protocol

1. In sterile conditions, harvest 2-4 mL of Escherichia coli BL21 cells with the fluorescent protein of interest (YFP, GFP, RFP, or ECFP) in a microcentrifuge tube
2. Centrifuge at 10,000 rpm for 1 minute between every milliliter harvested
3. Discard the supernatant after each centrifugation
4. After obtaining the desired pellet, add 1 mL of M63 medium supplemented with 1 mM MgSO4, 0.1 μM thiamine, and the corresponding antibiotic (Carbenicillin for YFP and GFP, Chloramphenicol for RFP, and Kanamycin for ECFP) to the microcentrifuge tube
5. Centrifuge at 10,000 rpm for 1 minute.
6. Discard the supernatant
7. Resuspend the pellet in 1 mL of supplemented M63 medium
8. Take 75 μL from each sample and place them in cells for optical density measurement, each with an additional 1425 μL of distilled water
9. Measure the optical density of each sample using only distilled water as the blank
10. After obtaining the optical density, multiply the value by 20 to determine the optical density of the samples obtained in step number 7
11. Use the formula C1V1 = C2V2 to determine how much volume to add from the samples in step 7 to obtain dilutions at different optical density values with M63 medium
12. Prepare the dilutions in new microcentrifuge tubes
13. Analyze the samples in the device for constructing the calibration curve