Team:ZJUT-China/Experiments/basic experiment
Human Practices
Basic experiments
Making competent cells
Materials
Protocols
Day 1
Streak the cells on a plate with LB agar and incubate at 37°C
Day 2
Preculturepick an isolated colony from the plate and inoculate 50ml of LB with it. Incubate overnight at 37°C shaking the culture at 200 rpm.
Day 3
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ All the manipulations of bacteria are carried out in a laminar flow cabinet.
✔ Work sufaces should be decontaminated with 70% alcohol.
✔ All materials that have been in touch with cells will be autoclaved.
Transformations
Materials
Protocols
1. Chill on ice an aliquot of 100μL of chemically competent cells
2. Add to 2-5μL of DNA to the aliquot
3. Incubate on ice for 30minutes
4. Incubate at 42°C for 90 seconds
5. Add 900μL of LB
6. Incubate at 37°C for 1 hour
7. Plate the cells with appropriate antibiotic
Safety considerations
Wearing laboratory coat, gloves is necessary to follow this procedure.
All the manipulations of bacteria are carried out in a laminar flow cabinet.
Work sufaces should be decontaminated with 70% alcohol.
All materials that have been in touch with cells will be autoclaved.
Bacterial glycerol stock
Materials
Protocols
1. Put 900μL of the cell culture into the cryovial and add 900μL of 30% glycerol. Vortex the mix.
2. Store in the -80°C freezer
Safety considerations
Wearing laboratory coat, gloves is necessary to follow this procedure.
All the manipulations of bacteria are carried out in a laminar flow cabinet.
Work sufaces should be decontaminated with 70% alcohol.
All materials that have been in touch with cells will be autoclaved.
Plasmid isolation
Materials
Overnight cell culture
CWBIO ® PurePlasmid Mini Kit
Protocols
1. Use 4-6mL of the cell culture and centrifuge at 13000 rpm for 30 seconds. Decant the supernatant into waste receptacle
2. Add 250μL buffer P1(with RNase A). Resuspend the cell pellet with vortex oscillator.
3. Add 250μL buffer P2. Mix gently by inverting the tube 4-6 times.
4. Add 350μL buffer P3. Mix gently by inverting the tube 4-6 times immediately.
5. Centrifuge at 13000 rpm for 5 minutes
6. Decant the supernatant into the Spin Columns DM and centrifuge at 13000 rpm for 30 seconds. Decant the flow-through into waste receptacle and place the spin column back.
7. Add 450μL buffer PB and centrifuge at 13000 rpm for 30 seconds. Decant the flow-through into waste receptacle
8. Add 400μL buffer PB and centrifuge at 13000 rpm for 1 minute. Decant the flow-through into waste receptacle
9. Place the spin column into a new collection tube and add 30μL buffer EB. Incubate at room temperature for 2 minutes and centrifuge at 13000 rpm for 1 minute. Store at -20°C. (Pre-heat the buffer EB to 65-70°C)
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ All materials that have been in touch with cells will be autoclaved.
PCR clean-up
Materials
Sample
CWBIO DNA Clean-up Kit
Protocols
1. Add 5 times volume of buffer PB and mix it up.
2. Put the Spin Columns DM into the collection tube. Add 200μL buffer PS into the spin column.
3. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle.
4. Add the solution acquired in step 1 into the spin column. Leave at room temperature for 1minute. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle.
5. Add 500μL buffer PW. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle.
6. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle. Leave at room temperature for several minutes.
7. Place the spin column into a new collection tube and add 30μL buffer EB. Incubate at room temperature for 2 minutes and centrifuge at 13000 rpm for 1 minute. Store at -20°C.
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ All materials that have been in touch with cells will be autoclaved.
Colony PCR
Materials
Protocols
1. Colonies are picked and pipetted down in 20μL of sterile PCR water. 2μL are used as template for the PCR.
2. On ice, add all components in a 250μL tube, making up to a 10μL volume reaction.
| components | Volume(μL) |
|---|---|
| Vazemy® green taq mix | 5 |
| Primers forward and reverse | 0.5 each |
| ddH2O | 3 |
| Template DNA | 1 |
3. Transfer the tubes to a PCR amplifier
4. Set the program.
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ Follow the instructions.
Overlap
Material
Protocols
1. On ice, add all components in a 250μL tube, making up to a 50μL volume reaction.
| components | Volume(μL) |
|---|---|
| Phanta Max Super-Fidelity DNA Polymerase | 1 |
| Primers forward and reverse | 2 each |
| ddH2O | up to 50 |
| Template DNA | x |
| 2× Phanta Max Buffer | 25 |
| dNTP mix | 1 |
2. Transfer the tubes to a PCR amplifier
3. Set the program.
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ Follow the instructions.
Agarose gel electrophoresis
Material
Electrophoresis comb
Electrophoresis tray
50mL beaker
erlenmeyer flask
UV transilluminator
Voltage source
Microwave
Agarose
TAE buffer
Vazemy® 10×Loading buffer
Molecular weight ladder
D-red nucleic acid dye
Protocols
Prepare agarose gel
1. Weight 1g of agarose powder in an erlenmeyer flask
2. Measure 100mL of TAE(1×) and add it to the erlenmeyer flask
3. Put the erlenmeyer flask in the microwave until the agarose is completely dissolved
4. Put 20mL of the mix into the beaker and add 4μL D-red nucleic acid dye and mix them
5. Set the comb in the tray and seal the mix inside
6. Let the mix cool down till it can be added to the electrophoresis tray
Running agarose gel
1. When agarose gel is solidified, remove the comb and cover the gel with TAE(1×)
2. Load the molecular weight ladder into the extreme well.
3. Add loading buffer to each DNA sample
4. Load the samples in the remaining wells
5. Run the gel with required voltage
6. Visualize the DNA fragments using a UV transilluminator
Safety considerations
✔ Some of nucleic acid dye is toxic. So we use one of the safe oneD-red.
✔ Used agarose gels are disposed in certain container.
✔ Wearing laboratory coat, gloves to follow this procedure.
DNA gel extraction
Material
Microcentrifuge
Heating block
Vortex mixer
cutter
CWBIO® gel extraction kit
Microcentrifuge tubes
Protocols
1. take a cutter to excise the DNA fragment from an agorse gel
2. determine the weight of the gel slice and transfer it to a microcentrifuge tube. For each 100mg of agarose gel add 100μL buffer PG.
3. Incubate the sample and vertex briefly until the gel slice is completely dissolved.
4. Put the Spin Columns DM into the collection tube. Add 200μL buffer PS into the spin column.
5. Add the solution acquired in step 3 into the spin column. Leave at room temperature for 1 minute. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle.
6. Add 450μL buffer PW. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle.
7. Repeat step 6
8. Centrifuge at 13000rpm for 1 minute. Decant the flow-through into waste receptacle. Leave at room temperature for several minutes.
9. Place the spin column into a new collection tube and add 30μL buffer EB. Incubate at room temperature for 2 minutes and centrifuge at 13000 rpm for 1 minute. Store at -20°C.
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ Follow the instructions.
One step clone
Material
250μL centrifuge
Insert DNA
Linearized plasmids
Vazemy® 5×CE II Buffer
Exnase II
1. On ice, add all components in a 250μL tube, making up to a 10μL volume reaction.
| components | Volume(μL) |
|---|---|
| Exnase II | 2 |
| ddH2O | Up to 20 |
| Vazemy® 5×CE II Buffer | 3 |
| Template DNA | x |
2. Gently mix it up
3. Leave it at 37°C for 30 minutes then chill it at 4°C.
Safety considerations
✔ Wearing laboratory coat, gloves is necessary to follow this procedure.
✔ Follow the instructions.
SDS page electrophoresis
Material
50ml beakers
Ice bucket
Ultrasonic cell crusher
Marker coomassie brilliant blue dye
Agarose gel
Electrophoresis tank, comb, tray
Voltage source
eStain L1
Gel imager
Protocol
1. Dilute PBS 10 times.Fill an ice bucket with ice. Chill four 50ml centrifuge tubes in ice.
2. Transfer our four different culture solution to four centrifuge tubes.Centrifuge at 12000rpm at 4 for 10 minutes. Decant the supernatant into waste receptacle.
3. Add 5ml PBS to each of the four centrifuge tubes. After mixing up, centrifuge at 12000rpm at 4 for 10 minutes. Finally, decant the supernatant of each tube into waste container.
4. Repeat 3.
5. Add 25ml PBS to each of the four centrifuge tubes and mix up. Put the four centrifuge tubes into four beakers which are all filled with ice.
6. Put the beakers in the ultrasonic cell crusher for 10 minutes.
7. Transfer the four solutions to four EP tubes. Centrifuge at 12000rpm at 4 for 10 minutes.
8. Transfer 0.075ml of each kind of supernatant to another four EP tubes.
9. Add 0.025ml coomassie brilliant blue dye to each of the four tubes. Put the tubes into boiled water for 10 minutes.
10. Pour fresh SDS-PAGE buffer into the electrophoresis tank. Set the comb in the tray.
11. Load the marker into the extreme well. Load the samples in the remaining wells.
12. Run the protein gel with required voltage for about 40 minutes.
13. Use the eStain L1 protein staining instrument for 10 minutes.
14. Visualize protein content using a Gel imager.
Safety considerations
✔ Laboratory coat, gloves are necessary.
✔ Marker coomassie brilliant blue dye is of great toxicity. Must be very cautious when manipulating.
✔ Used agarose gels are disposed in certain container.
✔ All the manipulations of bacteria are carried out in a laminar flow cabinet.
✔ All materials that have been in touch with cells will be autoclaved.
