Team:Tec-Monterrey/Experiments

Protocols

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Preparation and Management of Reagents of Speciality

Protocol 1.1 Calcium Competent Cells – Escherichia coli

Protocol 1.2 Calcium Competent Cells – Chromobacterium violaceum

Protocol 2. Transformation of Calcium Competent Cells

Protocol 3. Miniprep

Protocol 4.1. Electro-Competent Cells – Escherichia coli

Protocol 4.2. Electro-Competent Cells – Chromobacterium violaceum

Protocol 6. Digestion with Restriction Enzymes

Protocol 7. Electrophoresis

Protocol 8. Gel Extraction

Protocol 9. Ligation

Protocol 10. Expression and Purification of Proteins

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Preparation and Management of Reagents of Speciality

LB Broth Preparation

1. Add 20 g of LB to 1 L of distilled water.
2. Autoclave.

LB Broth Preparation

1. Add 20 g of LB and 15 g of Agar to 1 L of distilled water.
2. Autoclave.

Antibiotics and IPTG

1. Measure the desired amount of antibiotic for making a stock solution and add to ethanol or distilled water.
   1. Ampicillin = 100 mg/ml stock solution in 50 ml half volume of water and half volume of 100% ethanol.
   2. Chloramphenicol = 35 mg/ml in ethanol
   3. IPTG = 100 mM concentration
2. Filter with a 0.22 µm filter.

Solutions for Miniprep

• Solution I
  • Tris- Hcl 25mM
  • EDTA 10mM
• Solution II
  • NaOH 0.2 mM
  • SDS 1%
• Solution III
  • Sodium acetate 3M
  • Acetic Acid 2M

1. Solutions for Miniprep

2. Keep solution I and III at 4ºC and solution II at room temperature.

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Protocol 1.2 Calcium Competent Cells – Chromobacterium violaceum

Approximate time:

around 5 hours

Material

• 100 mM CaCl 2
• 200mL LB
• 4 sterile centrifuge tubes
• Overnight E. coli culture

Equipment & Apparatus

• Centrifuge.
• Spectrometer.
• Laminar flow hood
• Incubator with shaker

Previous steps

Autoclave 200mL of LB media placed in a 1L flask, glycerol, 4 centrifuge tubes, microcentrifuge tubes and 1L of 100 mM CaCl 2 .

Chill overnight at 4°C the 100 mM CaCl 2 .

Inoculate a 20mL starter culture of LB (no antibiotics). Grow culture at 37°C in shaker overnight.

Steps

1. Inoculate 200mL of LB media with approximately 10mL starter culture. Measure the OD600 to make sure it is around 0.1
2. Grow in 37°C shaker for around 1.5 to 2 hours until OD600 reaches 0.4-0.6. Immediately divide the culture in sterile centrifuge tubes (about 40 mL) in each tube and put them on ice. Chill the culture for 30 minutes.
3. Harvest the cells by centrifugation at 3000 g for 5 minutes at 4°C.
4. Decant the supernatant and gently resuspend each pellet in half volume of ice cold CaCl2.
5. Put the cells on ice and chill the culture for 20 minutes.
6. Harvest the cells by centrifugation at 3000 g for 5 minutes at 4°C.
7. Decant the supernatant and gently resuspend each pellet in half volume of ice cold CaCl2.
8. Put the cells on ice and chill the culture for 15 minutes.
9. Harvest the cells by centrifugation at 3000 g for 5 minutes at 4°C.
10. Decant the supernatant and gently resuspend the pellet of one of the tubes in 500μL of ice cold CaCl2 and 20% of glycerol. Repeat this step for all the pellets.

References

Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

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Protocol 1.1 Calcium Competent Cells – Escherichia coli

Approximate time:

around 5 hours

Material

• 100 mM CaCl 2
• 200mL LB
• 4 sterile centrifuge tubes
• Overnight E. coli culture

Equipment & Apparatus

• Centrifuge.
• Spectrometer.
• Laminar flow hood
• Incubator with shaker

Previous steps

Autoclave 200mL of LB media placed in a 1L flask, glycerol, 4 centrifuge tubes, microcentrifuge tubes and 1L of 100 mM CaCl 2 .

Chill overnight at 4°C the 100 mM CaCl 2 .

Inoculate a 20mL starter culture of LB (no antibiotics). Grow culture at 37°C in shaker overnight.

Steps

1. Inoculate 200mL of LB media with approximately 10mL starter culture. Measure the OD600 to make sure it is around 0.1
2. Grow in 37°C shaker for around 1.5 to 2 hours until OD600 reaches 0.4-0.6. Immediately divide the culture in sterile centrifuge tubes (about 40 mL) in each tube and put them on ice. Chill the culture for 30 minutes.
3. Harvest the cells by centrifugation at 3000 g for 5 minutes at 4°C.
4. Decant the supernatant and gently resuspend each pellet in half volume of ice cold CaCl2.
5. Put the cells on ice and chill the culture for 20 minutes.
6. Harvest the cells by centrifugation at 3000 g for 5 minutes at 4°C.
7. Decant the supernatant and gently resuspend each pellet in half volume of ice cold CaCl2.
8. Put the cells on ice and chill the culture for 15 minutes.
9. Harvest the cells by centrifugation at 3000 g for 5 minutes at 4°C.
10. Decant the supernatant and gently resuspend the pellet of one of the tubes in 500μL of ice cold CaCl2 and 20% of glycerol. Repeat this step for all the pellets.

References

We adapted protocol 1.1.

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Protocol 2. Transformation of Calcium Competent Cells

Approximate time:

90 monites

Material

• Liquid LB media
• Petri dishes

Equipment & Apparatus

• Water bath.
• Laminar flow hood
• Incubator with shaker

Previous steps

Autoclave LB media and LB-agar media.

Steps

1. Mix 1 to 5μl of DNA (usually 10pg to 100ng) into 50μL of competent cells in a microcentrifuge tube.
2. Place the competent cell/DNA mixture on ice for 20min.
3. Heat shock each transformation tube by placing the bottom of the tube into a 42°C water bath for 60 seconds (time varies depending on the competent cells you are using).
4. Put the tubes back on ice for 2 min.
5. Add 500μl LB media (without antibiotic) and grow in 37°C shaking incubator for 1 hour.
6. Plate 100 μl of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
7. . Incubate plates at 37°C overnight.

References

Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). “Basic Protocol 1: Transformation Using Calcium Chloride.” Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

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Protocol 3. Miniprep

Approximate time:

60 - 180 minutes

Material

• Solution I. (50 mM TRIS pH 8.0, 10 mM EDTA)
• Solution II (200 mM NaOH, 1% w/v SDS)
• Solution III (3 M potassium acetate, pH 5.5)
• EtOH 100%
• EtOH 70%
• ddh20

Equipment & Apparatus

• Nanodrop
• Centrifuge

Previous steps

Prepare starter culture cells by inoculating a 15mL starter culture of LB with the cells that have the desired plasmid (with the appropriate antibiotic). Grow culture at 37°C in shaker overnight

Steps

1. Centrifuge the starter culture.
2. Decant the supernatant and resuspend the pellet in about 1mL of LB media
3. Transfer the cells into a Eppendorf tube.
4. Centrifuge 14,000 rpm / 1 minute.
5. Decant the supernatant.
6. Add 200uL of solution I.
7. Use a pipette to resuspend the pellet.
8. Add 2uL of RNAse
9. Incubate 5min at room temperature
10. Add 200uL of Solution II
11. Mix gently by inversion 6 times
12. Incubate 5 min at room temperature
13. Add 200uL of Solution III
14. Mix gently by inversion 6 times.
15. Incubate 5 min on ice.
16. Centrifuge 14,000 rpm for 10 min.
17. Transfer the supernatant to a new tube (around 500uL).
18. Add 2 volumes cool of 100% EtOH (around 1mL).
19. Incubate at -20°C for 10 min (can be from 10 min to 2 hours).
20. Centrifuge 14,000 rpm for 10 min.
21. Decant the supernatant.
22. Add 200uL of cool 70% EtOH.
23. Use a pipette to resuspend the pellet.
24. Centrifuge 14,000 rpm for 5 min.
25. Decant the supernatant.
26. Dry pellet at 37 ° C for 5 min.
27. Add 30uL of ddH2O and resuspend pellet.
28. Measure DNA concentration in nanodrop.

References

Qiagen. (2009, November 20). Miniprep/Qiagen kit protocol. Retrieved June 2, 2014, from http://openwetware.org/wiki/Miniprep/Qiagen_kit_protocol

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Protocol 4.2. Electro-Competent Cells – Chromobacterium violaceum

Approximate time:

around 5 hours

Material

• LB Medium
• Sterile dH2O
• Glycerol

Equipment & Apparatus

• Spectrometer
• Centrifuge
• Laminar flow hood
• Incubator with shaker

Previous steps

Autoclave LB media placed in a 1L flask, glycerol, centrifuge tubes, microcentrifuge tubes and sterile dH2O. Chill overnight at 4°C water and glycerol. Inoculate a 12mL starter culture of LB (no antibiotics). Grow culture at 37°C in shaker overnight.

Steps

1. Inoculate 200mL of LB media with approximately 10mL starter culture. Measure the OD600 to make sure it is around 0.1.
2. Grow in 37°C shaker for around 1.5 to 2 hours until OD600 reaches 0.4-0.6.
3. Centrifuge at 3200 g for 12 minutes at 4°C
4. Decant the supernatant and gently resuspend each pellet in one volume of ice cold dH2O.
5. Centrifuge at 3200 g for 12 minutes at 4°C
6. Decant the supernatant and gently resuspend each pellet in one volume of ice cold dH2O with 10% glycerol.
7. Centrifuge at 3200 g for 12 minutes at 4°C
8. Decant the supernatant and gently resuspend each pellet in half volume of ice cold dH2O with 10% glycerol.
9. Centrifuge at 3200 g for 12 minutes at 4°C
10. Decant the supernatant and gently resuspend each pellet in 300 times concentrate of ice cold dH2O with 10% glycerol.
11. Aliquot 50μL into sterile 1.5 mL microfuge tubes and store at -80°C freezer.

References

Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

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Protocol 5.1. Transformation of Electro-Competent Cells – Escherichia coli

Approximate time:

around 2 hours

Material

• 1 mL, 100µL and 10µL Micropipettes
• SLB SOC media
• Petri Dishes
• Electroporation cells

Equipment & Apparatus

• Electroporator
• Laminar flow hood
• Incubator with shaker

Previous steps

Autoclave LB liquid media (500 µL per transformation) and LB-agar media (one control per antibiotic).

Steps

1. Keep a 1.7 ml centrifuge tube (previously incubated for 15 minutes at -20ºC) and a 2 mm cuvette on ice at all time.
2. Take 1 μl of DNA (ligation or plasmid) and gently mix with 50 μl of electrocompetent cells in the the cold 1.7 ml microcentrifuge tube.
3. Transfer all volume (51 μl) to the cuvette.
4. Pulse at 3000V (or 2500 if desired) 25 μF 200 ohms.
5. Immediately add 1 ml of SOC medium.
6. Transfer all volume to a 15 ml centrifuge tube and incubate 1 hour at 37oC 250
7. Incubate 2 plates of LB + antibiotic of interest, at 37ºC 20 minutes before electroporated cell’s incubation time ends.
8. Take 50 μl of electroporated cells and plate them in one of the LB plates.
9. Take 100 μl of electroporated cells and plate them also on the other plate.
10. Incubate at 37 ºC.

References

Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

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Protocol 6. Digestion with Restriction Enzymes

Approximate time:

2 hrs

Material

• PCR tubes
• DNA sample
• Restryction Enzymes (EcoRI, SpeI, PstI, XbaI)
• NEB Enzyme buffer
• Nuclease free dH2O

Equipment & Apparatus

• Thermocycler

Previous steps

Measure the DNA concentration of the sample using the Nanodrop.
Calculate the required concentrations of the components depending on the DNA of the sample, and the reaction volume.
Ideally you must put 1 enzyme unit per each µg of DNA, yet the provider recommends using 10 times that quantity and adjust the volumes. Our most typical reaction was set up to be 20 µL.

Steps

1. Set and label your PCR tubes.
2. With the micropipette first pour the nuclease free dH2O.
3. Pour the NEB buffer 10X to get the 1X concentration.
4. Put the DNA sample next, this sample must be as clean as possible.
5. The enzymes must be put lastly, be careful to handle them because they are very temperature sensitive. (Handle with ice and return them to the fridge as soon as possible).
6. Set up the Thermocycler at 37º for 1h and inactivate the enzyme at 80º for 20 mins.
7. Analyze using agarose electrophoresis.

References

Double Digest Protocol with Standard Restriction Enzymes https://www.neb.com/protocols/2014/05/07/double-digest-protocol-with-standard-restriction-enzymes
Optimizing Restriction Endonuclease Reactions https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
Digestion Protocol for BioBrick Assembly Kit (E0546) https://www.neb.com/protocols/1/01/01/digestion-protocol-e0546

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Protocol 7. Electrophoresis

Approximate time:

120 min

Material

• Agarose
• TAE Buffer
• Loading dye
• 10 kb and 1 kb weight stair
• Ethidium bromide
• Pipettes and tips
• DNA sample

Equipment & Apparatus

• Electrophoresis chamber
• Voltage source
• Transiluminator

Previous steps

Digestion. Usually this protocol precedes the electrophoresis.
Prepare TAE stock solution (50X).

Steps

Prepare the gel

1. The amount of agarose depends on the size of the DNA.
2. Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer. The volume in the lab gel box is 40mL.
3. Microwave until the agarose is fully melted and cool down a few minutes.
4. Pour the agarose solution into the taped gel box. Carefully pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.

Load the gel

1. Mix the DNA samples with 5X loading dye solution to make it reach 1X. (Except for 1 µL of sample you can use 1µL of Loading dye).
2. Pour the mixture in the wells. Be careful with the bubbles and breaking the gel with the pipette.
3. Fill the chamber with TAE and run the gel 70 min at 90 V and ~180 mA
Stain and visualize the gel

1. Stain the gel soaking it in EtBr, be careful not to break it.
2. Reveal using the transiluminator, first focus using the white light lamp and use the UV to make the bands visible.

References

Agarose Gel Electrophoresis http://www.addgene.org/plasmid-protocols/gel-electrophoresis/

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Protocol 8. Gel Extraction

Approximate time:

60 min

Material

• Eppendorf tubes
• Nuclease free dH2O

Equipment & Apparatus

• Water bath
• Microcentrifuge

Previous steps

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

Steps

1. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
2. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel.
3. Add 1 gel volume isopropanol to the sample and mix.
4. Place a QIAquick spin column in a provided 2 ml collection and apply the sample to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
5. To wash, add 750 μl Buffer PE to QIAquick column, let the column stand 2–5 min after addition of Buffer PE and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
6. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
7. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 20 μl water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, let the column stand for 5 min, and then centrifuge for 1 min.

References

QIAquick Gel Extraction Kit. https://www.qiagen.com/us/shop/sample-technologies/dna/qiaquick-gel-extraction-kit/#orderinginformation

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Protocol 9. Ligation

Approximate time:

60 min

Material

• DNA sample
• T7 ligase
• 10X T7 Buffer
• Nuclease free dH2O

Equipment & Apparatus

• Thermocycler

Previous steps

Measure the DNA concentration of the sample using the Nanodrop.
Calculate the required concentrations of the components depending on the DNA of the sample, and the reaction volume.
Ideally you must put 1 enzyme unit per each µg of DNA, yet the provider recommends using 10 times that quantity and adjust the volumes. Our most typical reaction was set up to be 20 µL.

Steps

1. Set up the reaction in a microcentrifuge tube on ice.
   

   Component	20 μl Reaction
   T4 DNA Ligase Buffer (10X)	2 μl
   Vector DNA (4 kb)	50 ng (0.020 pmol)
   Insert DNA (1 kb)	37.5 ng (0.060 pmol)
   Nuclease-free water	to 20 μl

2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. Incubate at 16°C overnight at the Thermocycler.
4. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
5. Analyze using agarose electrophoresis.

References

Ligation Protocol with T4 DNA Ligase. https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

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Protocol 10. Expression and Purification of Proteins

Expression and Purification of Proteins

around 7 hours

Material

• LB media
• IPTG 100 mM (or any other inducer)
• Overnight transformed cell culture
• Antibiotic

Equipment & Apparatus

• Centrifuge
• Spectrometer
• Laminar flow hood
• Incubator with shaker

Previous steps

Prepare lysis buffer depending on the desired protein to be purified. Prepare a flask of 500 mL with 200 mL of LB medium.

Steps

1. Inoculate 100 mL of LB media with antibiotic and with starter culture and measure the OD600 to make sure it is around 0.1
2. Grow in 37°C shaker at 250 rpm for around 1.5 to 2 hours until OD600 reaches 0.4-0.6.3. Change medium and add again antibiotic and make induction by adding IPTG in order to be diluted to between 0.1 mM to 1 mM.
3. Change medium and add again antibiotic and make induction by adding IPTG in order to be diluted to between 0.1 mM to 1 mM.
4. Grow in 30°C shaker at 100 rpm for around 3 to 4 hours.
5. Transfer into sterile centrifuge tubes and harvest the cells by centrifugation at 3000 g for 10 minutes at 4°C.
6. Add 5 mL of lysis buffer and gently resuspend.
7. Make sonification while having the tubes in ice for 3 min (10 seconds of sonification and 10 seconds of waiting).
8. Centrifuge tubes and harvest the cells by centrifugation at 3000 g for 10 minutes at 4°C.
9. Keep supernatant.

References

Protocol was given by one of our advisors.

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