Team:Worldshaper-Wuhan/Protocol

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Protocol

Design the primer

Obtain the target gene fragment

Obtain the enzyme cutting vector

Ligation

Transformation

Cell culture

Examination

Protocol

1. Design the primer

2. Obtain the target gene fragment

a. Run PCR(50ul)

3. Obtain the enzyme cutting vector

a. Enzyme digestion

I. add 1-2ul enzyme, 2ul DNA fragments, 5ul 10x buffer into the tube; II. centrifuging and shake it to make it equally distribution; III. seal it; IV. put the into a 37 ° C water bath and digested for 4 hours; V. Remove the tube from the water bath, recycle the gel and put it at 4° C refrigeration.

b. Agar Gel electrophoresis c. Gel recycling

4. Ligation

Meterial: recombinase, the enzyme cutting vector, the target gene fragment and H2O I. Add DNA fragments and cleaved plasmid as 4:1 into test tube; II. Add recombinase and water; III. Place it into the 37 ° C water bath for 30 min.

5. Transformation (shake bacteria, coated plate, pick bacteria, elution)

a. Making a medium

I. Mix(250ml)

II. Mix the solution with a magnetic stirrer; III. Distribute the stirred solution into 40 test tubes and sterilize them with autoclave for 30 min; IV. Remove the solution, store some under 4 ° C and add others with agarose powder and store at 4 ° C after it solidify.

b. Transformation

I. Take the medium from 4 ° C;

II. Add the plasmid into the competent cell. Ice for 30 min, place in 42 ° C for 90 seconds and - Ice again for 2 min;

III. Place the test tube on a shaker at 37 ° C and 45 rpm for 1 hour;

IV. Take the competent bacteria from the shaker;

V. Swab the competent bacteria on the medium;

VI. Immerse sticks into alcohol twice;

VII. After the stick cooled down, use it to spread the bacteria by drawing ‘z’ shape on the medium;

VIII. After that, seal the medium, mark the corresponding date and bacteria, culture the medium in the incubator overnight at 37 ° C ;

c. Pick the bacteria

I. Remove the cultured dish from incubator;

II. Use the tip of the gun to spot the bacteria to remove the bacteria;

III. Throw the tip that has the bacteria into liquid medium and put it into the shake at 150 rpm overnight;

IV. Extract the cultured bacteria from the cultured medium.

d. obtain the plasmid

I. Add 500 μl of equilibration solution BL to the adsorption column CP3, centrifuge for 1 minute at 12000 rpm, discard the waste from the collection tube, and return the adsorption column to the collection tube;

II. Take 1-5 ml of bacterial solution, add to the centrifuge tube, centrifuge 1 minute at 12000 rpm, try to remove the supernatant;

III. Add 250 μl of solution P1 to the centrifuge tube where the bacteria are left to precipitate, use the gun to stir until no precipitate can be observed by naked eye;

IV. Add 250 μl of P2 to the centrifuge tube, gently flip the tube 6-8 times. So that the cell lysis;

V. Add 350 μl to the centrifuge tube P3, immediately gently flip the tube up and down 6-8 times until fully mixed, this time there will be white flocculent precipitation;

VI. Transfer the supernatant collected from the previous step to the suction column P3 and place it in the collection tube (do not draw the precipitate);

VII. Use 12000rpm to centrifuge 30-60 seconds, drained the waste in the collection tube, place the adsorption column into the collection tube;

VIII. Add 600 μl of rinse PW solution to the adsorption column, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste in the collection tube, and place the column into the collection tube;

IX. Repeat step 7;

X. Centrifuge adsorption column CP3 again at 12,000 rpm for 2 minutes, drained the waste;

XI. Place the adsorption column in a clean centrifuge tube, add 50-100 μl of the elution buffer to the middle of the adsorbent membrane, allow the mixture to stand for 2 minutes at room temperature, centrifuge at 12000 rpm for 2 minutes and collect the plasmid solution into the centrifuge tube;

e. Ligation

Material: recombinase, the enzyme cutting vector, the target gene fragment and H2O

I. Add DNA fragments and cleaved plasmid as 4:1 into test tube;

II. Add recombinase and water. Place it into the 37 ° C water bath for 30 min;

f. Sequencing

II. Send the plasmid for sequencing after observing the line;

cell culture

1. Culture Conditions

I. Atmosphere: CO2, 5%;

II. Temperature: 37 °C.

2. Complete Growth Medium

I. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

3. Transfection

I. Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 60~70% confluency at the time of transfection;

II. For plasmid DNA transfection experiment, we recommend using 4ug DNA per well in a 24-well plate;

III. Add 4ug of plasmid DNA to the 400ul transfection medium opti, and mix immediately. Incubate the transfection mixture for 2 minutes at room temperature;

IV. Add 8ul of transfection reagent to the 400ul transfection medium opti, and mix immediately. Incubate the transfection mixture for 5 minutes at room temperature;

V. Allow the transfection Reagent/DNA complex to incubate at room temperature for 20 minutes to let transfection complex form;

VI. Add the transfection complex to the cells drop wise;

VII. Replace fresh medium after 4~6 hours and continue to culture for 24~48 hours.

Examination

1. fluorescence microscopy

2. measure the value of GFP fluorescence by plate reader