Protocol
1. Obtain the part of MALAT1 fragment by PCR
a. Run PCR(50ul)
b. Set extension step at 1 min per kilobase of product;
c. Initial denaturation for 3mins at 94℃;
d. Anneal primers for 30s ;
e. Extend DNA for 1min at 72℃;
f. Repeat step V-VII for 30 cycles.
g. Final extension for 5 mins at 72℃;
h. Run 2ul on a gel to check size at concentration of PCR product.
2. Obtain the recombinant pCDNA3-MCS-nPepper plasmids
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.
d. 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 T4 DNA ligase and water;
III. Place it into the 37 ° C water bath for 30 min.
e. Transformation (shake bacteria, coated plate, pick bacteria, elution)
I. Making a medium 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.
f. 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 ;
g. 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.
h. 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;
i. 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;
g. Sequencing
Send the plasmid for sequencing.
3. Plasmid transfections
a. Cell culture conditions
Atmosphere: CO2, 5%; Temperature: 37 °C.
b. Complete Growth Medium
To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
c. Transfection
I. The Hela cells were resuscitated prior to the transfection
II. The transfection was performed only when the cell density was 60%–80%，then mixing different volumes of microRNA-145/ microRNA-22（final volumes：2 μg、1 μg、0.5 μg、0 μg）, 2μg plasmid DNA (miR-145-sponge /miR-22-sponge) and transfection reagent (Lipo3000).
III. The transfection complexes were added to the pre-cultured density-appropriate Hela cell
IV. Sample s were harvested after 48 hours. Then we add 2μm HBC fluorescent dye into per well, and incubation for 2 hours.
V. Detect fluorescence under a fluorescence microscope
Transfection Schematic in 24 well Plate
