CONTENTS

1.  Preparation of liquid LB medium

2.  PCR- Amplifying virus gene

3.  Agarose gel Electrophoresis

4.  Inoculation

5.  Plasmid Extraction

6.  DNA Gel Extraction

7.  Double enzyme digestion and homologous recombination

8.  Ligation and homologous recombination

9.  Transformation-Heat Shock

10.  IPTG-induced protein expression

11.  Ultrasonication and protein purification

12.  SDS-PAGE protein electrophoresis

 

1. Preparation of liquid LB medium

Material:

Reagent	Mass (g)
Tryptone	10
Yeast extract	5
NaCl	10

Add ddH₂O to 1 L, sterilization at 121 ℃ for 20 min.

Solid plus 1.5% agar.

 

Apparatus:

Test tube	1
triangular flask	1
measuring cylinder (0-500 mL)	1
ultra-clean bench	1
media dispenser	1
balance	1
Pipette (100-1000 μL)	1
Petri dishes	8
sealing strips	8

Procedure: On this basis, add 100 μL of antibiotics (diluted by 1000 times), prepare the plate after the temperature is not hot, write the relevant information on the bottom with a marker, and then pour it into the plate while the medium is not solidified, wait for solidification and then send to the refrigerator (during the period, disinfect the bottleneck, do not take your hands out of the workbench).

 

2.  PCR- Amplifying virus gene (GII.4-VP1 + GII.17-VP1 + RV VP7 gene)

Reagent	Volume (μL)
Mix	25
Template	1
Forward primer	1
Reverse primer	1
ddH2O	22
Total	50

 

PCR Procedures:

Step	Temperature ( ℃)	Time
1	98	3 min
2	98	30 sec
3	60	30 sec
4	72	2 min
5	72	10 min
6	4	forever

PS: Steps 2 to 4 cycle 30 times.

 

Apparatus:

PCR tubes	10
pipettes (range: 2  μL-20   μL)	2
pipettes (range: 20  μL-20 0  μL)	2
PCR Thermal Cycler	1

 

3.  Agarose gel Electrophoresis

(1)  Preparation of TAE

Material:

Reagent	Volume/mass
TAE buffer powder	1 g
ddH₂O	500 mL

 

(2)  Preparation of Agarose gel

Reagent	Volume/mass
Agarose	1 g
1x TAE(buffer)	100 mL(1%)
GelRed	2-3 mL
Marker	6 μL

Apparatus:

Electrophoresis instrument	1
UV analyzer	1
measuring cylinder	1
balance	1
Erlenmeyer flask	1
Mold (base plates and tooth combs)	1
Pipettes (2-20μL)	2

 

Procedure:

a. Weigh out 1 g of agarose.

b. Pour 100 mL TAE into a measuring cylinder.

c. Mix them into an Erlenmeyer flask and put in the powder before the liquid.

d. Microwave the mixture on high heat for 2 min.

e. Prepare the mold, put in the base plate and tooth comb, and then add the mixture. Wait for it to solidify.

f. Add the Marker into the first well.

g. After that, close the lid, connect the positive and negative poles, and let stand for 20 min.

h. Remove the prepared gel and place it in the UV analyzer.

i. The Marker position on the left corresponds from bottom to top to a DNA length.

 

 4. Inoculation:

Procedure:

1. Sterilize both hands and equipment brought into the clean bench.

2. Label an EP tube with the correct data, subject, and experimenter name.

3. Disinfecting tubes and equipment using an alcohol burner.

4. Add 3 mL LB liquid medium into the labeled tube.

5. Add 20 μL glycerol bacteria (pGEX-4T-1. etc.) into the labeled tube.

6. Add 3 μ L Ampicillin (100 mg/mL) into the labeled tube.

7. Close the labeled tube with its cap.

8. Sterilized all the equipment.

9. Wash both hands with proper detergents.

10.culture at 37 ℃, 220 rpm in constant temperature shaker for 12~18h.

 

5. Plasmid Extraction

Material:

reagent	Volume/mass
overnight cultured bacterial solution	1.5-5 mL
Buffer SP1	250 μL
Buffer SP2	250 μL
Buffer SP3	350 μL
Buffer DW1	500 μL
Wash solution	500 μL
Elution Buffer	50-100 μL

Apparatus:

centrifuge	1
pellets	6
adsorption columns	6
collection tubes	12
Pipette (range: 0.5 μL ~ 10 μL)	2
Pipette (range: 20  μL ~ 200  μL )	2
Pipette (range: 100  μL ~ 10 00  μL )	2

Procedure:

a. Take 1.5-5 mL of overnight cultured bacterial solution, centrifuge at 8000 xg for 2 min to collect bacteria, and discard the medium.

b. Add 250 μL of Buffer SP1 to the pellet and suspend the bacteria thoroughly.

c. Add 250 μL of Buffer SP2 and immediately mix and invert the centrifuge tube 5-10 times gently and let stand at room temperature for 2-4 min (lysing the bacteria).

d. Add 350 μL of Buffer SP3 and immediately mix and invert the tube 5-10 times gently (Neutralization, Plasmids are preferentially adsorbed on the membrane).

e. Centrifuge at 12000 xg for 5-10 min, pipette the supernatant into the adsorption column, centrifuge at 8,000 xg for 30 s ec , and drain the liquid in the collection tube.

f. Add 500 μL Buffer DW1, centrifuge at 9000 xg for 30 s ec , and drain the liquid from the collection tube.

g. Add 500 μL Wash solution, centrifuge at 9000 xg for 30 sec, and drain the liquid from the collection tube (Repeat once) (wash away the unwanted substances).

h. Centrifuged the empty adsorption column at 9000 xg for 1 min (Drain the cleaning solution).

i. Place the adsorption column into a 1.5 mL centrifuge tube and add 50-100 μL Elution Buffer in the center of the adsorption membrane, let it stand at room temperature for 1 min followed by centrifugation for 1 min, then preserve the plasmid liquid in the collection tube.

 

6. DNA Gel Extraction

Material:

reagent	Volume/mass
DNA	40 μL
Buffer B2	300 μL
Wash solution	500 μL
Elution Buffer	50-100 μL

Apparatus:

centrifuge	1
adsorption columns	6
collection tubes	6
Pipette (range: 20  μL ~ 200  μL )	2
Pipette (range: 100  μL ~ 10 00  μL )	2
1.5 mL centrifuge tubes	6

Procedure :

a.  Add 300 μL of buffer B2 into the centrifuge tubes with the solution that finished PCR.

b.  The liquid was moved into the adsorption columns and centrifuged at 8000 xg for 30 sec to dump the liquid from the collection tubes.

c.  Add 500 μL Wash solution, centrifuge at 9000 xg for 30 sec, and drain the liquid from the collection tubes (Repeat once).

d.  Centrifuged the empty adsorption column at 9000 xg for 1 min.

e.  Place the adsorption columns into 1.5 mL centrifuge tubes and add 50-100 μL Elution Buffer in the center of the adsorption membrane, let it stand at room temperature for 1 min followed by centrifugation for 1 min, then preserve the plasmid liquid in the collection tubes.

7. Double enzyme digestion

Reagent	Volume(μL)
Plasmid  ( pGEX-4T-1 )	30
EcoRI	1
XhoI	1
Buffer (Cutsmart)	5
ddH2O	13
Total	50

Reagent	Volume(μL)
DNA (GII.4- VP1 /GII.17- VP1 )	20
EcoRI	1
XhoI	1
buffer(Cutsmart)	5
ddH2O	23
Total	50

 

Procedure:

1. Add all of the elements in the form into a 1.5 mL tube using pipettes.

2. Heat in the water bath at 37 ℃ for 30 min.

 Apparatus:

pipette (range: 100 μL- 1000 μL)	1
Water bath	1

 

8.  Ligation and homologous recombination

I.  Ligation

Apparatus:

PCR tubes	4
pipette (range: 2  μL- 20  μL)	1
electrophoresis instrument	1
UV analyzer	1
PCR Thermal Cycler	1

Materials:

Reagent	Volume ( μL )
pGEX-4T-1	4
GII.4- VP1 /GII.17- VP1	13
T4 ligase	1
T4 ligase buffer	2
Total	20

Procedure:

a.  Add all of the elements in the form into PCR tubes using pipettes.

b.  Adjust the PCR profile to 16 °C for 30 min with 65 °C for  5 min and start running it.

c.  After the gel is set, demould, pour TAE that can overwhelm the gel into the electrophoresis instrument, inject the prepared DNA solution into the wells one by one with a pipette (except the first well), and then inject DNA Marker into the first well.

d.  After that, close the lid, connect the positive and negative poles, and let stand for 20 min.

e.  Remove the prepared gel and place it in the UV analyzer.

 

II. homologous recombination

Reagent	Volume ( μL )
pGEX-4T-1	3
RV -VP7	1
GII.4-VP1/GII.17-VP1	1
2x Clon Express Mix	5
Total	10

 

Procedure:

a.  Add the above reagents to a 1.5 mL centrifuge tube. (Note: the DNA in the second row should be added separately to the plasmid in the first row).

b.  Place in a water bath at 50 °C for 15 min.

9.  Transformation-Heat Shock

 Materials:

Reagent	Volume(μL)
competent cell(DH5α/BL21/Nissle 1917)	50
Plasmid(pGEX-GII.4-VP1/pGEX-GII.17-VP1 pGEX-RV-GII.4-VP1/pGEX-RV-GII.17-VP1)	5
LB liquid medium	950

Apparatus:

centrifuge	1
Pipette (range: 100 μL ~ 10 00 μL )	2
microtubes	6
Icebox	1
Water bath	1
shaker	1

Procedure:

a.  Add 50 μL competent cell (DH5α/BL21/Nissle 1917) and 5 μL plasmid into the microtube.

b.  Leave the tube on ice for 30 min .

c.  Transfer the tube to the water bath at 42 ℃ for 90 sec.

d.  Transfer the tube onto ice for 5 min.

e.  For each tube, add LB medium in it until the total volume reaches 1 mL.

f.  Place the tube in the thermostatic shaker at 37 ℃, 220 rpm for 30 min.

g.  Centrifuge the tube at 9000 xg for 1 min, then discard 950 μL of the liquid and mix the content left in the tube

h.  Place Petri dishes upside down in an incubator at 37 °C overnight.

 

10.  IPTG-induced protein expression

a.  Expanded the culture seed solution: 1% Inoculated volume is transferred to 100 mL LB Liquid medium, and OD₆₀₀ is grown at 37 °C to 0.4-0.6 (about 3 to 4 h).

b.  Performed IPTG-induced expression BL21(DE3). The final concentration of 0.25 mM IPTG was added to induce expression.

c.  Overnight culture was performed at 16 °C, 220 rpm in incubator.

 

11. U ltrasonication and purif ication

Material:

Reagent	Volume/mass
Bacteria liquid medium	90 mL
Lysis buffer(lysozyme)	Depend on the steps
BeyoGold GST-tag Purification Resin	Depend on the steps
cracking buffer	0.5 mL
elution buffer	9 mL
eluent buffer	0.5 mL each time
GSH	30 mg, 1 mL

Apparatus:

centrifuge	1
balance	1
Pipettes (100 μL-1000 μL)	3
Ice container	1
ultrasonic crusher	1
EP tubes	8
affinity column	1
refrigerator	1
centrifuge tubes	8

 

Procedure:

a. Divide three bottles of E. coli BL21, 30 mL each.

b. Remove the supernatant after centrifugation.

c. Remove the weight of the bottle and measure the wet weight of the bacteria.

d. Configuration of the lysozyme is 100 mg/mL (23 mg + 230 μL Lysis buffer).

e. On the basis of 0.1 g sediment weight/1000 μL of lysozyme and lysis buffer are added together, blown well, and put into a container of ice water for ultrasonic crushing.

f. Ultrasonic cracking bacteria on ice, ultrasonic power 40%, each ultrasonic treatment 5 sec, each interval 5 sec, a total of 3 ultrasonic treatments.

g. The bacterial solution after three tubes of ultrasonic crushing was added to 8 EP tubes of 1 mL respectively.

h. Centrifuge at 4 °C at 10000 xg for 20-30 min.

i. Take the supernatant and merge it together.

j. Extract 1 mL BeyoGold GST-tag Purification Resin, centrifuge 1000 xg at 4 °C for 10 sec, and discard the storage solution.

k. Add 0.5 mL of cracking buffer and centrifuge at 4 °C, and repeat the steps.

l. Put in the supernatant, and place in a 4 °C shaker for 60 min.

m. The mixture of the lysate and BcyoGold GST-tag Purification Resin was loaded into the empty column tube of the affinity column.

n. The lid at the bottom of the purification column was opened, and the liquid in the column was discharged under the action of gravity. About 20 mL  of the flow fluid was collected for subsequent analysis.

o. Wash the column twice and add 0.5 mL of lysis buffer each time, collect, and put in the refrigerator.

p. To configure the elution buffer, first add 30 mg GSH and 1 mL of elution buffer. Next, 9 mL of elution buffer and 1 mL of GSH were mixed at a ratio of 9: 1.

q. Wash the column twice, each time with 0.5 mL eluent buffer, each eluent is collected into a different centrifuge tube. The eluent obtained from the collection is the purified GST label protein sample, which is finally placed in the refrigerator.

 

12. SDS-PAGE protein electrophoresis

Material:

Reagent	Volume/mass
glue solution (2x)	2.7 mL
lower layer of glue-grade flushing solution (2x)	2.7 mL
water	--
upper glue solution (2x)	0.75 µL
the color upper glue buffer (2x)	0.75 µL
modified coagulant	70 µL
electrophoresis buffer	--

 

Apparatus:

glue molds	2
glue mixing cups	4
Pipettes (100 μL-1000 μL)	1
glass plates	4
Pipette (20 μL-200 μL)	1
Pipette (2 μL-20 μL)	1
Pipette (0.1 μL-2.5 μL)	1

Procedure:

I.  SDS-PAGE modified acrylamide color gel.

1.  Assemble the glue molds and configure the lower layer of glue 1.00 mm.

2.  Take the same volume of the lower layer of glue solution (2 x ) and the lower layer of glue grade flushing solution (2 x ) 2.7 mL each, and mix them in the glue mixing cup.

3. Add 55 µL of the modified coagulant to the mixture in Step 2 and stir gently to avoid bubbles.

4. Add the lower layer of glue solution mixed in step 3 to the glue maker, so that the liquid level is 1.5 cm away from the upper edge of the glass plate, and then cover the lower layer of glue solution with a layer of water to keep the gel surface flat.

5. Stand at room temperature (25 ℃) for 6-10 min, and when the boundary between the lower layer and the covering phase appears, the gelatin is said to have solidified.

6. Configure the upper layer glue 1.00 mm.

7. Slowly pour off the covering phase, take an equal volume of the upper glue solution (2x) and the color upper glue buffer (2x), 0.75 µL each, and mix well in a new glue cup.

8. Add 15 microliters of modified coagulant to the mixture in step 7 and stir gently to avoid bubbles.

9. Add the upper glue solution to the upper layer of the lower glue until the gel solution reaches the top of the glass plate. Slowly insert the comb into the gel to avoid bubbles.

10. Stand for 10~15 min, wait for the upper layer of amine to solidify, carefully pull out the comb, use the gun head or syringe to absorb the electrophoresis buffer, rinse the sample hole, and then perform SDS-PAGE electrophoresis.

 

II. Protein glue staining and decolorization

Material:

Coomassie bright blue dye

Decolorizing agent :

Reagent	Volume ( μL )
Ethanoic acid	10%
dd water	50%
Alcohol	4 0%

Apparatus:

Plastic box	1
Horizontal shaker	1

 

III. Dyeing and decolorization of protein glue

Procedure:

1.  Soak the protein glue in Coomassie bright blue dye for 1 hour.

2.  Decolorize the stained protein by using the decolorizing agent 3 times, each time lasting for 10 min .