Protocols

Experiment name: polymerase chain reaction experiment

Reagents:

Reagent	quantity
2 × Phanta Max Master Mix	25 μl
dNTP mix	1 μl
Taq DNA polymerase	1 μl
Forward primer	2 μl
Reverse primer	2 μl
Template DNA	5 μl (less than 1/10 of the total volume)
ddH2O	14 μl (up to 50 μl)

Protocol:

1.Add 25 µl of buffer solution tube into a 0.1ml EP tube.

2.Add 1 µl of dNTP mix into the EP tube.

3.Add 1 µl of Taq DNA enzyme into a 0.1 ml EP tube.

4.Add 2 µl each of forward and reverse primers to the mixture.

5.Add 5μ of template DNA.

6.Add ddH2O (double-distilled water) to a total volume of 50 μl.

7.Vortex the mixture to ensure that it is well-mixed.

8.Centrifuge the mixture to ensure that all components are well-mixed.

Experiment name: agarose gel electrophoresis

Reagent	quantity
agarose gel powder	0.6 g (1:100 ratio of agarose gel powder to TAE buffer)
TAE buffer	60 ml
Nucleic acid dye “GoldView”	3 μl
10 × DNA loading buffer	5 μl

Protocol:

1.Wash the conical flask to be used.

2.Dry your weighing scoop with a paper towel.

3.Take out a piece of weighing paper and fold it in half along the diagonal.

4.Place the weighing paper on an electronic scale, tare the paper, and weigh 0.6 grams of agarose gel powder (the ratio of agarose gel powder to TAE buffer is 1:100).

5.Pour the weighed agarose gel powder into a conical flask.

6.Use a cleaned measuring cylinder to measure 60 ml of TAE buffer.

7.Pour the measured TAE buffer into a conical flask and mix thoroughly with the agarose gel powder.

8.Heat the mixture in a microwave oven until the agarose is completely dissolved.

9.Place the conical flask under running water for about 20 seconds to allow it to cool to approximately 40 ℃.

10.Add 3 µl of the nucleic acid dye GoldView to the solution.

11.Gently shake the conical flask to distribute the dye evenly.

12.Carefully pour the solution onto the horizontal plate and put on the comb to make wells.

13.After the gel has completely solidified, gently pull out the comb.

14.Place the gel into the electrophoresis tank and add TAE to submerge the gel.

15.Add 5 μl of 10 × DNA loading into each sample and mix it.

16.Add the samples into the wells with a pipette gun.

17.Close the lid of the electrophoresis tank, turn on the power, and perform electrophoresis for about 10~15 minutes. Stop before DNA loading reaches the end of the gel.

Experiment name: Agarose gel recycling

Reagent	quantity
PC buffer	100 μl of PC buffer per 0.1 g of agarose gel
BL buffer	500 µl
PW buffer	600 µl
EB buffer	43 µl

Protocol:

1.Add 500 µl of BL buffer into a CB2 absorption column.

2.Centrifuge at 12,000 rounds per minute (rpm) for 1 minute.

3.Discard the liquid.

4.Cut out the gel containing our desired DNA fragment and put it into an EP tube.

5.Add 100 μl of PC buffer per 0.1 g of agarose gel.

6.Heat the EP tube in a metal bath at 50 ℃ for approximately 15 minutes to dissolve the agarose gel.

7.Transfer the solution into the CB2 collection column.

8.Centrifuge at 12,000 rpm for 1 minute.

9.Add 600 μl of wash buffer PW.

10.Centrifuge at 12,000 rpm for 1 minute.

11.Repeat steps 9 and 10.

12.Place the CB2 collection column in a collection tube.

13.Centrifuge at 12,000 rpm for 2 minutes.

14.Take out the CB2 collection column and place it in the air for several minutes until completely dried.

15.Put the CB2 collection column in a clean 1.5 ml EP tube and add some ddH2O.

16.Place at room temperature for 2 minutes.

17.Centrifuge at 12,000 rpm for 2 minutes to collect the DNA fragments.

Experiment name: enzyme digestion experiment

Sal1-SS/FD	1 µl
BamHI-SS/FD	1 µl
Buffer-10 × SS	5 µl
10 × DNA loading	5 µl
DNA sample	43 µl
Plasmid backbone	4 µg
ddH2O	up to 50 µl (for plasmid)

Protocol:

1.Add 1 µl of each of the Sal1-SS/FD, 1 µl of BamHI-SS/FD, and 5 µl of Buffer-10 × SS into the EP tube containing DNA obtained through agarose gel recycling.

2.Add 1 µl of each of the Sal1-SS/FD, 1 µl of BamHI-SS/FD, and 5 µl of Buffer-10 × SS into the EP tube containing the plasmid backbone.

3.Place the EP tubes in a metal bath at 37 ℃ for 30-35 minutes.

4.Repeat the agarose gel electrophoresis experiment and the agarose gel recycling experiment.

Experiment name: plasmid ligation experiment

Reagent	quantity
Target fragment	4 µl
Plasmid backbone	1 µl
Ligase enzyme	5 µl

Protocol:

1.Add 1 µl of plasmid backbone and 4 µl of target fragment into an EP tube.

2.Add 5 µl of ligase enzyme into the EP tube.

3.Put the EP tube into the PCR machine at 16 ℃ for 1 hour.

Experiment name: plasmid transformation

Reagent	quantity
Plasmid	10 µl
DH5α sensory state bacteria cells	40 µl
LB medium with ampicillin antibiotics	600 µl
LB agar plate	1

Protocol:

1.Add the ligation products into the EP tube containing DH5α competent cells in the ultra-clean bench.

2.Put the EP tube on ice for 30 minutes.

3.Heat the EP tube in a dry bath at 42 ℃ for 90 seconds.

4.Put the EP tube on ice for 2 minutes.

5.Add 600 µl of LB medium into the EP tube.

6.Place the EP tube in a shaker incubator at 37 ℃, 200 rpm for 50 minutes.

7.Centrifuge the EP tube at 4,000 rpm for 3 minutes to collect the DH5α competent cells.

8.Discard 500 µl of the supernatant fluid.

9.Use a pipette to resuspend the cells.

10.Transfer the mixture onto the LB agar plate with ampicillin antibiotics.

11.Spread the cells evenly on the LB agar plate.

12.Place the LB agar plate in an incubator at 37 ℃ for 12-14 hours.

Experiment name: The preparation of LB Medium and single bacterial colony selection

Reagent	quantity
yeast extract	1 g
tryptone	1 g
NaCl	0.5 g
water	100 ml
Agar (solid LB medium)	1.5 g
antibiotic ampicillin	8 µl ( 1:1000 )

Protocol:

1.Add 1 g of yeast extract, 1 g of tryptone, 0.5 g of NaCl, and 100 ml of water into a conical flask.

2.The LB medium is autoclaved.

3.Add 100 µl of ampicillin antibiotic into the LB medium for solid LB medium.

4.Pick out a small bacterial colony from the last experiment and put it into a test tube and put it into the LB medium.

5.Place the test tube in a shaker incubator at 37 ℃, 200 rpm for 12-14 hours.

Experiment name: plasmid extraction

Reagent	quantity
P1 buffer	250 µl
P2 buffer	250 µl
P3 buffer	350 µl
BL buffer	500 µl
PW buffer	600 µl
EB buffer	100 µl
LB medium	8-10 ml

Protocol:

1.Centrifuge at 12,000 rpm for 1 minute to collect the bacterium in 1.5 ml EP tube and pour out the supernatant.

2.Add 250 µl of P1 buffer and shake to mix the solution evenly.

3.Add 250 µl of P2 buffer and gently turn to mix the solution evenly.

4.Add 350 µl of P3 buffer and gently turn to mix the solution evenly.

5.Centrifuge the 1.5 ml EP tube at 12,000 rpm for 10 minutes.

6.Add 500 µl of BL buffer into a CP3 collection column at 12,000 rpm for 1 minute.

7.Discard the liquid.

8.Transfer the supernatant from the fifth step into a CP3 collection tube.

9.Centrifuge the CP3 collection tube at 12,000 rpm for 1 minute.

10.Discard the liquid.

11.Add 600 µl of PW buffer into the CP3 collection tube.

12.Centrifuge the CP3 collection tube at 12,000 rpm for 1 minute.

13.Discard the liquid.

14.Repeat steps 12 and 13

15.Centrifuge at 12,000 rpm for 2 minutes.

16.Take out the CP3 collection column and place it in the air for several minutes until completely dried.

17.Put the CP3 collection tube into a new 1.5 ml EP tube.

18.Add 50 µl of ddH2O.

19.Place under room temperature for 2 minutes.

20.Centrifuge the EP tube at 12,000 rpm for 2 minutes.

Experiment name: the resuscitation of normal gastric cell line GES-1; gastric cancer cell line AGS and gastric cancer cell line MGC-803

Reagent	quantity
GES-1	1 ml
AGS	1ml
MGC-803	1 ml
1640 medium	4 ml

Protocol:

1.Put the frozen cells in a water bath at 37 ℃.

2.Shake the tubes until there is no more ice.

3.Centrifuge at 1,000 rpm for 3 minutes.

4.Discard the supernatant.

5.Add 1 ml of 1640 medium to resuspend the cells.

6.Transfer the cells into a Petri dish.

7.Place the cells in carbon dioxide (CO2) incubator at 37 ℃ for 24 hours℃.

Experiment name: Cell passaging

Reagent	quantity
PBS buffer	3 ml
trypsin	500 µl
1640 medium	500 µl

Protocol:

For all three cell lines:

1.Discard the medium.

2.Wash the cells using 1ml PBS solution each time for 2 times.

3.Digest the cells on the petri dish using 500 µl trypsin for 2 minutes.

4.Add 500 µl of 1640 medium to terminate the digestion.

5.Transfer the cells into a centrifuge tube.

6.Centrifuge at 1,000 rpm for 3 minutes.

7.Discard the supernatant.

8.Add 1ml of 1640 medium to resuspend the cells.

9.Transfer the cells into a new petri dish.

10.Place the cells in the carbon dioxide (CO2) incubator at 37 ℃ for 24 hours.

Experiment name: cell transfection

Reagent	quantity
PBS	3 ml
trypsin	1 ml
1640	6 ml
Opti-MEM	100 µl
Plasmid DNA	2 µg

Protocol:

For all three cell lines:

1.Discard the old medium.

2.Add 3 ml of PBS solution to wash the cells.

3.Add 1 ml of trypsin to digest the cells for 2 minutes.

4.Stop digestion by adding 1 ml of 1640 medium.

5.Resuspend the cells using a pipette.

6.Transfer the cells into centrifuge tubes.

7.Centrifuge at 1000 rpm for 3min.

8.Discard the supernatant.

9.Add 2 ml of 1640 medium into the centrifuge tube.

10.Resuspend the cells using a pipette.

11.Put the medium containing the cells into the 6-well and the 24-well cell culture plates.

12.Place the cells in the carbon dioxide (CO2) incubator at 37 ℃ for 24 hours.

13.Dilute the 2 µg of plasmid DNA with 100 µl of non-serum diluent Opti-MEM.

14.Add 2 µl of Neofect transfection reagent.

15.Leave them for 15 minutes and place them into the 6-well and the 24-well cell culture plates.

Experiment name: Bioluminescence

Reagent	quantity
5× cell lysate	500µl
PBS	2ml
Firefly luciferase substrate	20 µl
supernatant	20 µl
Renilla luciferase substrate	20 µl

Protocol:

1.500 µl of 5× cell lysate diluted with 2 ml PBS on ice.

2.24 wells, PBS wash cells 1 time, 500 μl of each well.

3.Add 100 µl of lysate and shake on a shaker for 10 min.

4.Transfer the lysate into a 1.5 ml EP tube, 12000 rpm, 10min.

5.dispense 20 of firefly luciferase substrate placed in a 1.5 ml EP tube.

6.Set up a double Perspex luciferase reporter.

7.Zeroing with an empty 1.5 ml EP tube.

8.Add 20 µl of supernatant to 20 μl firefly luciferase substrate, blowing 20 times, to ensure that each frequency, step, and blowing strength are consistent.

9.Add 20 µl Renilla luciferase substrate after the test, and then click ok to measure.

10.Calculate the ratio of Filevli Luciferase/Renilla Lucilas per tube.

Experiment name: RNA extraction

Protocol:

1.Sample preparation. Organizational sample: Cut small pieces of liver tissue, add 1 ml RNAiso Plus lysis buffer, grind the liver tissue thoroughly with a homogenizer, and aspirate the upper layer of liquid into a 1.5 ml EP tube. Cell sample: trypsin digestion for 3 minutes, terminate digestion, centrifuge to remove supernatant, wash once with PBS buffer, discard supernatant, and add 500 µl RNAiso Plus lysis reagent.

2. Extraction. Add 200 µl of trichloromethane to every 1 ml of lysis buffer in the fume hood. Thoroughly shake and mix well, let it stand for 5 minutes, and obvious layering can be seen. Centrifuge at 12000 rpm at 4 ℃ for 15 minutes.

3.Sedimentation. Take the supernatant and place it in a new EP tube. Add the same volume of isopropanol, mix gently, and let it settle on ice for 10 minutes. Then centrifuge at 12000 rpm at 4 ℃ for 10 minutes.

4. Wash. Pour out the supernatant, add 500 µl of ethanol wash solution, invert and mix well, centrifuge at 4 ℃ and 8000 rpm for 5 minutes, and pour out the supernatant. Wash again.

5. Remove the ethanol from the tube and let it sit for about 5 minutes. The white precipitate will turn into a transparent gelatinous substance.

6. Add an appropriate volume of DEPC water and mix gently. The obtained RNA is immediately reverse transcribed or frozen at -80℃.

Experiment name: Reverse Transcription

Protocol:

1.Except for DNA. Take 2 µl RNA as a template for reverse transcription, add 4 µl RNA Free Water, 2 µl gDNA, gently mix, and incubate at 42℃ for 2 minutes.

2. Reverse transcription. Add 2 µl of reverse transcriptase, gently mix well, incubate at 50 ℃ for 15 minutes, at 85 ℃ for 2 minutes, and then cool down to 4 ℃ to obtain the reverse transcribed cDNA.

Experiment name: quantitative PCR

Protocol:

1.Prepare a mixture, with each reaction system as shown in the table below.

Master mix	5 µl
Rox	0.2 µl
Primer F	0.4 µl
Primer R	0.4 µl
cDNA	1 µl
RNase-Free H ₂ O	3 µl
TOTAL	10 µl

2.Reaction program, set according to the table below.

Temperature	time	Number of cycles
95 ℃	5 minutes	1
95 ℃	30 seconds	40
60 ℃	30 seconds	40
72 ℃	30 seconds	40
72 ℃	3 minutes	1

Experiment name：Fluorescence value of EGFP measurement

Protocol：

1.Full wavelength fluorescence detector

2.Place the cell sample on the fluorescence measuring instrument rack

3.Select 488nm excitation light wavelength

4.Click to run measurement