Team:Shanghai Metropolis/Experiments

Experiments

PCR

· Materials

Reagent Volume/mass
pUC57-VP1 150ng
pUC57-LTB 150ng
PrimeSTAR Max Premix 75μL
VP1-FP (10μM) 5μL
VP1-RP (10μM) 2.5μL
VP1-Lkr-RP (10μM) 2.5μL
LTB-FP (10μM) 2.5μL
LTB-RP (10μM) 2.5μL
ddH2O 30μL
*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.
Apparatus VAmount
PCR thermal cycler 1
Pipette (range: 2μL-20μL) 1
Pipette (range: 20μL-100μL) 1
Micro-centrifuge tube 3

· Procedure

  1. Add 20μL pUC57-VP1 into 2 micro-centrifuge tubes with each tube 10μL. Label these tubes with VP1 and VP1-lkr. Then add 10μL pUC57-LTB into another micro-centrifuge tube. Label this tube with LTB. Use the pipette (range: 2μL-20μL) to transfer the liquid above.

  2. Add VP1-FP and VP1-RP into VP1. Add VP1-FP and VP1-Lkr-RP into VP1-Lkr. Add LTB-FP and LTB-RP into LTB. Use the pipette (range: 2μL-20μL) to transfer the liquid above.

  3. Add 25μL PrimeSTAR Max to each micro-centrifuge tubes by using a pipette (range: 20μL-100μL). Add 10μL ddH2O to each micro-centrifuge tubes by using a pipette (range: 2μL-20μL).

  4. Shake three micro-centrifuge tubes and then insert them into the PCR Thermal Cycler. Use the following setting:

Process Temperature Time
Denaturation 98℃ 10s
Annealing 65℃ 15s
Extension 72℃ 5s
Back to step 1 (35X)
Final elongation 72℃ 5mins
-- 12℃ ∞

Overlap Extension PCR (OE PCR)

· Materials

Reagent Amount used Final concentration
PrimeSTAR Max Premix (2×) 25μL 1×
VP1-FP 10 ~ 15pmol 0.2 ~ 0.3μM
LTB-RP 10 ~ 15pmol 0.2 ~ 0.3μM
ddH2O To 50μL --
Template (VP1-Lkr & LTB fragment) 150ng + 150ng --
*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.
Apparatus Amount
Micro-centrifuge tube (1.5ml) 3
Pipette (range: 0.5 μl ~ 10 μl) 1
Pipette (range: 20 μl ~ 200 μl) 1
Pipette tips (of all three sizes) 6
Conical centrifuge tube (15 ml) 1
Micro-centrifuge tube rack 2
PCR Thermal Cycler 1

· Procedure

  1. Set up a PCR system (50μL).

  2. Place the micro-centrifuge tube holding the system into the PCR Thermal Cycler.

  3. Set up a reaction program.

Process Temperature Time
Denaturation 98℃ 10s
Annealing 65℃ 15s
Extension 72℃ 5s
Back to step 1 (35X)
Final elongation 72℃ 5mins
-- 12℃ ∞

  1. Start the PCR reaction.

  2. After the reaction, take out the micro-centrifuge tubes from the PCR thermal cycler.

  3. Run a DNA gel electrophoresis and extract the DNA fragments from the gel.

Colony PCR

· Materials

Reagent Amount used Final concentration
PrimeSTAR Max Premix (2×) 10μL 1×
Forward primer 10 ~ 15pmol 0.2 ~ 0.3μM
Reverse primer 10 ~ 15pmol 0.2 ~ 0.3μM
ddH2O To 50μL --
Template (incubated ATCC 334 chemically competent cells with plasmids pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB) 1μL --
*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.
Apparatus Amount
Micro-centrifuge tube (1.5ml) 3
Pipette (range: 0.5 μl ~ 10 μl) 1
Pipette tips (of all three sizes) 6
Conical centrifuge tube (15 ml) 1
Micro-centrifuge tube rack 2
PCR Thermal Cycler 1

· Procedure

  1. Set up a PCR system (20μL).

  2. Place the micro-centrifuge tube holding the system into the PCR Thermal Cycler.

  3. Set up a reaction program.

Process Temperature Time
Denaturation 98℃ 10s
Annealing 65℃ 15s
Extension 72℃ 5s
Back to step 1 (35X)
Final elongation 72℃ 5mins
-- 12℃ ∞

  1. Start the PCR reaction.

  2. After the reaction, take out the micro-centrifuge tubes from the PCR thermal cycler.

  3. Run a DNA gel electrophoresis.

Agarose Gel Electrophoresis

· Materials

Reagents Volume/mass
Agarose 1g
TAE Buffer ~500mL
Ts-GelRed 10μL (10000X)
DNA Loading Buffer 10X in template
Marker 5μL
Template --
Apparatus Amount
Conical flask (range: 300mL, division value: 100mL) 1
Pipette (range: 2μL-20μL) 1
Pipette (range: 0.5μL-10μL) 1
Weighing paper 1 piece
Electronic balance 1
Spatula 1
Microwave oven 1
Aluminum foil 1 piece
Mold for creating gel 1
Electrophoresis chamber 1
UV transilluminator 1
Scalpel 1

· Procedure

Making gel

  1. Place the weighing paper on the electronic balance, weigh 1g agarose, and pour it into the conical flask.

  2. Pour 100mL TAE into the conical flask and shake it.

  3. Cover the conical flask with the aluminum foil and place it into the microwave oven for 3 minutes, until the granules of agarose are well dissolved and invisible.

  4. Use the pipette (range: 2μL-20μL) to transfer 10μL Ts-GelRed into the conical flask.

  5. Pour the gel into the mould and insert a ‘comb’ into the gel. Wait for 10 minutes until the gel is solidified.

Electrophoresis

  1. Pour about 400mL TAE into the electrophoresis chamber.

  2. Carefully take out the gel and replace it in the electrophoresis chamber.

  3. Add the loading buffer, 1/10 of the volume of the template, into the template by using the pipette (range: 2μL-20μL) and shake it.

  4. Use the pipette (range: 0.5μL-10μL) to add 5μL marker in the first hole of the gel, then add 5μL of each template into the following holes.

  5. Turn on the power of the electrophoresis chamber and wait for about 40 minutes (the specific time should depend on the markers we use).

Authentication and recycle

  1. Take out the gel and place it on the UV tray. The UV tray will then be placed into the UV transilluminator.

  2. Open the Image Lab and scan the gel.

  3. Comparing with the marker reference, determine the target section which will be extracted during the recycling process.

  4. Use the scalpel to cut the target section of the gel.

DNA Gel Extraction

· Materials

Reagents Volume/mass
Binding buffer (NTI) 200μL
Agarose gel 100mg
Buffer EB 25μL
Wash Buffer (NT3) 700μL
Apparatus Amount
Micro-centrifuge tube (1.5mL) 2
Thermostat 1
Spin column 1
Centrifuge 1

· Procedure

Gel extraction

  1. Add 100mg agarose gel and 200μL NT1 into one micro-centrifuge tube.

  2. Place the tube in the thermostat at 50°C for 5-10 minutes.

  3. Vortex the tube after ensuring all gel dissolves.

Binding DNA

  1. Insert a spin column into a collection tube and transfer the solution into the spin column.

  2. Centrifuge the tube at 12000rpm, 25°C for 1 minute.

  3. Discard the waste liquid from the collection tube.

Wash silica membrane

  1. Add 700μL NT3 solution into the spin column.

  2. Centrifuge the tube at 12000rpm, 25°C for 1 minute.

  3. Discard the waste liquid from the collection tube.

  4. Repeat steps 1-3 for 2 times.

Dry silica membrane

  1. Centrifuge the tube at 12000rpm, 25°C for 2 minutes

  2. Discard the waste liquid from the collection tube.

Elution

  1. Insert the spin column into a new micro-centrifuge tube.

  2. Add 25μL buffer EB to the center of the column membrane and incubate the column at room temperature for 2 minutes.

  3. Centrifuge the tube at 12000rpm, 25°C for 1 minute.

Restriction Enzyme Double Digestion

· Materials

Reagents Volume
VP1 fragment 10μL
VP1-LTB fragment 10μL
ddH2O 14μL
FD buffer 4μL
SalI enzyme 1μL
BamHI enzyme 1μL
Apparatus Amount
Micro-centrifuge tube 2
Pipette (range: 2μL-20μL) 2
Pipette (range: 0.5μL-10μL) 2
Thermostat 2

· Procedure

  1. Add 10μL fragments (VP1/VP1-LTB), 14μL ddH2O and 2μL buffer into one micro-centrifuge tube with a pipette(range:2μL-20μL)

  2. Add 0.5μL SalI and 0.5μLBamHI into the micro-centrifuge tube with another pipette (range:0.5μL-10μL)

  3. Place the micro-centrifuge tube in the thermostat at 37℃ for 30 minutes.

*The exact time and temperature should depend on the template

ClonExpress ligation reaction

· Materials

Reagents Volume
Ligation buffer 10μL
T4 ligase 10μL
VP1 fragment 20μL
VP1-LTB fragment 20μL
Vector (digested pGEX-6P-1) 30μL
ddH2O 10μL
Apparatus Amount
Micro-centrifuge tube（1.5 mL） 2
Pipettes (range:5-50μL） 1
Thermostat 1

· Procedure

  1. Add 20μL VP1 fragments to one micro-centrifuge tube and add 20μL VP1-LTB fragments to another tube.

  2. Add 5μL ligation buffer, 5μL T4 ligase, 15μL digested pGEX-6P-1, and 5μL ddH2O to each tube.

  3. Place the mixed reagents in the thermostat at 22 ℃ for 1 hour.

E. coli Transformation

· Materials

Reagents Volume
pGEX-6P-1-VP1 1μL
pGEX-6P-1-VP1-LTB 1μL
DH5α/BL21 chemically competent cell 100μL
Liquid LB medium (amp-) 500μL
Solid LB medium (amp+) 500μL
Apparatus Amount
Thermostat 1
Petri dish 2
Shaker 1
Pipette (Range: 0.5-2.5μL) 1
Pipette (Range: 20-200μL) 1
Conical centrifuge tube (1.5 mL) 2
Incubator 1
Bacterial culture tube 2

· Procedure

Transformation

  1. Thaw 100μL competent cells on ice for 1-2 minutes.

  2. Mix the cells with 1μL plasmids (pGEX-6P-1-VP1 or pGEX-6P-1-VP1-LTB) in one bacterial culture tube with one pipette (range: 0.5-2.5μL).

  3. Place the tubes in ice for 30 minutes.

Heat shock

  1. Place the tubes in the thermostat at 42℃ for 60-90 seconds.

  2. Place the tubes in ice bath immediately and cool them for 2-3 minutes.

Resuscitation

  1. Add 500μL LB medium (amp-) to every bacterial culture tube.

  2. Mix the solution uniformly, and incubate it in the shaker at 37℃,180rpm for 45-60 minutes.

Incubation

  1. Aspirate the transformed competent cells with one pipette (range: 20-200μL)

  2. Coat them uniformly on the LB solid media (amp+)

  3. Place the petri dishes at room temperature until the solution is absorbed.

  4. Turn the dishes upside down and incubate the cells at 37℃ for 12-16 hours.

L.paracasei Transformation

· Materials

Reagents Volume
ddH2O 300μL
20% glycerinum solution 400μL
MRS medium (amp-) 1800μL
MRS solid medium (amp+) 1800μL
ATCC334 Chemically Competent Cell 200μL
pGEX-6P-1-VP1 10μL
pGEX-6P-1-VP1-LTB 10μL
Apparatus Amount
Micro-centrifuge tube (1.5ml) 2
Centrifuge 1
Electroporation cuvette 2
Electroporation machine 1
Shaker 1
Incubator 1
Petri dish 2
Pipette (Range: 0.5-2.5μL) 1
Pipette (Range: 20-200μL) 1
Bacterial culture tube 2

· Procedure

Thaw

  1. Take out the competent cells from the refrigerator at -80℃ and thaw them at room temperature.

  2. Add 150μL ddH2O to one micro-centrifuge tube.

  3. Place it at room temperature for 20 minutes.

  4. Precool the centrifuge at 4℃.

  5. Centrifuge the tube at 13,000rmp, 4℃ for 3 minutes.

  6. Discard the supernatant.

Resuscitation

  1. Add 100μL precooled 20% glycerinum solution to the micro-centrifuge tube.

  2. Centrifuge the tube at 13,000 rpm for 10 minutes.

  3. Discard the supernatant.

Resuspension

  1. Add 100μL 20% glycerinum solution and 10μL pGEX-6p-1-VP1 or pGEX-6p-1-VP1-LTB to the centrifuge tube.

  2. Transfer the resuspension into electroporation cuvette with one pipette (range: 0.5-2.5μL).

Electroporation

  1. Set up the electroporation machine: 2000V, 25μF, 400Ω.

  2. Insert the cuvette into the machine respectively, and pulse one time.

Resuscitation

  1. Aspirate the resuspension from the electroporation cuvettes with one pipette (range: 0.5-2.5μL).

  2. Turn it into 900μL MRS media in a bacterial culture tube.

  3. Resuscitate the bacteria in the shaker at 37℃ for 3-4 hours.

Incubation

  1. Aspirate 100μL resuspension with another pipette (range: 20-200μL)

  2. Coat the resuspension on the MRS medium (amp+)

  3. Place the resuspension in the incubator at 37℃ for 36-72 hours.

Plasmid extraction (TIANprep Mini Plasmid Kit)

· Materials

Reagents Volume
Buffer BL 500μL
Buffer P1 250μL
Buffer P2 250μL
Buffer P3 350μL
Buffer PW 600μL
Buffer EB 50-100μL
Bacteria --
Apparatus Amount
Spin column CP3 1
Collection tube (1.5mL) 1
Micro-centrifuge tube 2
Centrifuge 1
Vortex machine 1

· Procedure

Column Equilibrium

  1. Insert one spin column CP3 into the collection tube.

  2. Add 500μL buffer BL to the tube.

  3. Centrifuge the tube at 12,000rpm for 1 minute.

  4. Discard the waste liquor in the collection tube.

  5. Insert the spin column back to it.

Bacteria Extraction

  1. Harvest 1-5mL overnight cultured (12 - 16h) bacteria into a centrifuge tube.

  2. Centrifuge the tube at 12,000rmp for 1 minute.

  3. Discard the supernatant.

  4. Invert the tube on the absorbent paper to dry.

Resuspension

  1. Add 250μL buffer P1 to the centrifuge tube.

  2. Insert the tube into the vortex machine until the bacteria are suspended thoroughly.

Lysis

  1. Add 250μL buffer P2 to the centrifuge tube.

  2. Turn it upside down for 6-8 times temperately.

Precipitation

  1. Add 350μL buffer P3 to the centrifuge tube.

  2. Immediately turn it upside down for 6-8 times temperately until the solution is uniformly mixed.

  3. Centrifuge the tube at 12,000rpm for 10min.

Transfer

  1. Aspirate the supernatant and transfer it to spin column CP3 in the collection tube.

  2. Centrifuge it at 12,000rpm for 30-60 seconds.

  3. Discard the waste liquor in the collection tube.

Rinse

  1. Add 600μL buffer PW to the spin column CP3.

  2. Centrifuge it at 12,000rpm for 30-60 seconds.

  3. Discord the waste liquor in the collection tube.

  4. Insert the spin column back to the collection tube.

  5. Repeat procedure 1-4.

  6. Centrifuge the tube at 12,000rpm for 2 min.

Elusion

  1. Insert the spin column CP3 into a clean micro-centrifuge tube.

  2. Add 50-100μL buffer EB to the center of the spin membrane.

  3. Place the tube in room temperature for 2 minutes.

  4. Centrifuge it at 12,000rpm for 2 minutes.

  5. Turn the plasmid solution to the centrifuge tube.

SDS-PAGE electrophoresis

· Materials

Reagents Volume
Liquid LB medium (amp-) 30mL
Ampicillin 300μL
ddH2O 1200μL
Loading buffer 300μL
Running buffer --
Marker 10μL
IPTG 75μL
SurePAGE (15%BT) 1 piece
Apparatus Amount
Pipette (range: 0.5-2.5μL) 1
Pipette (range: 20-200μL) 1
Pipette (range: 5mL) 1
Micro-centrifuge tube(1.5mL) 6
Electrophoresis chamber 1
Bacterial culture tube 6

· Procedure

  1. Transfer 400μL bacteria in each tube to six micro-centrifuge tubes with one pipette (range: 5mL).

  2. Centrifuge the six centrifuge tubes at 12,000rmp for 1 minute.

  3. Discard the supernatant.

  4. Add 200μL ddH2O and 50μL loading buffer to each tube.

  5. Insert the prepared gel (SurePAGE 15%BT) into the electrophoresis chamber.

  6. Add running buffer into the chamber until the gel is submerged.

  7. Add 10μL marker to the first hole of the gel and add 10μL bacterial solution in each tube into the following holes with one pipette (range: 2-20μL).

  8. Set up the electrophoresis chamber: 100V, 2h.

  9. Start the program.

Coomassie brilliant blue staining

· Materials

Reagents Volume
Coomassie brilliant blue staining solution --
Gel with protein after SDS-PAGE 1 piece
Apparatus Amount
Electrophoresis chamber 1
Box (1dm×1dm×0.5dm) 1
Microwave oven 1
Shaker 1

· Procedure

(It is conducted after SDS-PAGE)

  1. Discard the running buffer and take out the gel from the chamber.

  2. Transfer the gel to a box and add Coomassie Brilliant Blue to it until the gel is submerged.

  3. Heat up the gel in the microwave oven.

  4. Place the box on the shaker for 10 minutes.

  5. Take out the gel from the box and use Image Lab to scan the gel.

Western Blot

· Materials

Reagents Volume
Rabbit Anti-GST antibody --
Rabbit-HRP --
TBST solution --
ECL chemilumninecent liquid --
5% defatted milk solution --
BeyoECL Plus solution --
Gel with protein after SDS-PAGE 1 piece
Apparatus Amount
Trans-blot transfer 1
PVDF membrane 1
Shaker 1
Box(1dm×1dm×0.5dm) 1

· Procedure

(It is conducted after SDS-PAGE)

Transfer

  1. Submerge trans-blot transfer in the ice bath.

  2. Set the trans-blot transfer at 300mA.

  3. Place the gel and PVDF membrane in the trans-blot transfer for 60 minutes.

Blocking

  1. Turn the gel to a box, add TBST solution into it, and rinse the gel for 1-2 minutes.

  2. Discard the TBST solution.

  3. Add 5% defatted milk into the box until the gel is submerged.

  4. Place the box on the shaker at room temperature for 60 minutes.

Primary antibody incubation

  1. Discard the milk in the box.

  2. Add Rabbit Anti-GST antibody solution to the box.

  3. Place the box on the shaker at room temperature for 2 hours.

  4. Recycle the rabbit Rabbit Anti-GST antibody solution.

  5. Rinse the gel with TBST solution 3 times, 15 minutes for each time.

Secondary antibody incubation

  1. Discard the TBST solution in the box.

  2. Add Rabbit-HRP solution to the box.

  3. Place the box on the shaker at room temperature for 60 minutes.

  4. Recycle the Rabbit-HRP solution.

  5. Rinse the gel with TBST solution 3 times, 15 minutes for each time.

Detection of proteins

  1. Add BeyoECL Plus solution into the box.

  2. Use the software ImageLab to scan the gel.

Email us at: zhixuansong1022@qq.com; 1956461994@qq.com Search‘EV71terminator’ in Wechat to find more about us