Remove culture medium from the dish. Gently add 1 ml of phosphate buffered saline (PBS) to the dish, and shake it several times.
Remove PBS from the dish. Add 1 ml of pre-warmed 0.1% Trypsin to the dish. Gently shake it to get complete coverage of the cell layer. Incubate the cells at room temperature for 40-60 seconds. (Note: The actual incubation time varies with
the cell line used.)
Add 2ml of pre-warmed complete growth medium (DMEM with 10% FBS). Disperse the cells by pipetting over the cell layer surface several times.
Transfer the cells to 15 ml centrifuge tubes and centrifuge at 1200 ×g for 3 minutes.
Resuspend the cell pellet in 1-2 ml of pre-warmed medium and take 10 μl of medium for cell counting.
Cells were seeded in plates at the recommended density, and pipe the rest of the cells into new cell culture dishes, and put dishes back to the incubator (37°C, 5% CO2).
Dilute 2.5 ng of plasmids in 0.2 ml Opti-MEM® medium. Dilute 7.5µl of transfection reagent (LipofectaminTM 2000) in 0.2 ml Opti-MEM® medium and wait for 5 minutes.
Mix the plasmids and transfection reagent and wait for 20 minutes.
Take out the seeded the cells in 6-well plates from incubator. (Note: The number of cells is generally suitable at 70-80% confluence.) Remove the original complete growth medium (DMEM with 10% FBS), and then add 1.6 ml of the mixture of
the plasmid, transfection reagent and serum free medium to each well.
Remove culture medium from the dish. Gently add 1-2 ml of phosphate buffered saline (PBS) to the dish, and shake it several times.
Remove PBS from the dish. Add 1 ml of pre-warmed 0.1% Trypsin to the dish. Gently shake it to get complete coverage of the cell layer. Incubate the cells at room temperature for 40-60 seconds.
Add 2ml of pre-warmed complete growth medium (DMEM with 10% FBS). Disperse the cells by pipetting over the cell layer surface several times.
Transfer the cells to a centrifuge tube and centrifuge cells at 1200 ×g for 3 minutes.
Resuspend the cell pellet in 1-2 ml of pre-warmed medium and take 10 μl of medium for cell counting.
Put the small chamber into a 24-well plate, Transfer 30000-50000 cells to each small chamber. Add the 500 µl of DMEM medium in the 24-well plate, culture overnight.
Remove the medium from the small chamber with cotton balls (or remove it with a pipette, avoid destroying the film).
Take out the small chambers and remove the medium in the wells. Add 500 µl of PFA (multi chlorine formaldehyde) into the wells, and then put the small chambers back and let it sit for 30 minutes.
Take out the small chambers, remove the residual PFA in the wells by cotton swabs and add 500 µl of PBS to each well.
Add 500 µl of crystal violet dye to the wells and put back the small chambers (avoid making any bubbles). Stand it for 20 min for dyeing.
Wipe out the extra crystal violet dye on the film with a cotton swab, and count the number of cells under the microscope.
Prepare freezing medium and store at 2℃ to 8℃ until use.
Remove culture medium from the dish. Gently add 1 ml of phosphate buffered saline (PBS) to the dish, and shake it several times.
Remove PBS from the dish. Add 1 ml of pre-warmed 0.1% Trypsin to the dish. Gently shake it to get complete coverage of the cell layer. Incubate the cells at room temperature for 40-60 seconds. (Note: The actual incubation time varies with the cell line used.)
Add 2ml of pre-warmed complete growth medium (DMEM with 10% FBS). Disperse the cells by pipetting over the cell layer surface several times.
Centrifuge the cell suspension at 1200×g for 3 minutes. Aseptically decant supernatant without disturbing the cell pellets.
Resuspend the cell pellet in cold freezing medium.
Dispense aliquots of the cells into cryovials. Place the cryovials in an isopropanol chamber in order to decrease the temperature gradually and store them at -80℃ overnight.
Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen.
Centrifuge the cell suspension at 500×g for 5 minutes.
Determine the total number of cells and percent of viability by the automated cell counter. (If necessary, add growth medium to the cells to achieve the desired cell concentration and recount the cells.) Then collected 2×105~2×106 cells for assay.
Add pre-warmed PBS to the cells, centrifuge at 500×g for 5 minutes and then collect the cells.
Add 100 µl of pre-warmed Annexin V Binding Buffer to the cells and resuspend the cells.
Add 5 µl of Annexin V-FITC and 5µl of PI to the cells, mix them.
Protect the cells from light for 15 minutes at room temperature (20°C-25°C).
Add 400 µl of pre-warmed Annexin Binding Buffer to the cells, mix them, place the sample on ice and protect it from light.
Detect the results by flow cytometer within an hour.
Harvest the cells by centrifuging the overnight LB culture at 3000×g for 15 minutes. Remove all supernatant.
Add 250 µl of Resuspension Buffer (RB) to the pellet and resuspend cells until homogeneous. (Note: If cells were resuspended in a 15 ml centrifuge tube, and then transfer the cells in fresh EP tubes.)
Add 250µl of Lysis Buffer (LB). Mix gently by inverting the capped tube until that the lysate mixture is thoroughly homogenous. Incubate at room temperature for 5 minutes.
Add 250 µl of Neutralization Buffer (NB) and mix immediately by inverting the tube. (Note: Do not allow lysis and to proceed for more than 5 minutes.)
Centrifuge the lysate at 12,000 ×g for 5 minutes at room temperature. (Note: If the pellet does not adhere to the bottom of the tube, centrifuge the tubes at 12,000×g for another 5 minutes at room temperature for completely precipitated.)
Load the supernatant from cell lysate onto the equilibrated column.
Add 650 µl of Wash Buffer (WB) and centrifuge the column at 12000 ×g for 1 minute. Discard the flow-through.
Repeat step 7.
Centrifuge the column at 12000×g for 2 minute to completely remove the residual WB.
Add 30-50µl of Elution Buffer (EB) or water to the column to dissolve the DNA. Centrifuge the column at 12000 ×g for 1 minute.
Repeat step 10.
Store the purified DNA at 4°C for immediate use or aliquot the DNA and store at -20°C for long-term storage.
Amplified the DNA fragments by PCR with the system of 10xBuffer 10µl, dNTPs 200µmol/L, Upstream/downstream primer 10 µmol/L, Template DNA 2ng, Taq DNA polymerase 2.5µl, Mg2+ 1.5mmol/L and dd-water 30µl. PCR under the following conditions: 95°C for 30 seconds, 60°C for 1 minute and 68°C for 1 minute for 30 cycles.
Add 0.5µl (20ng) of PCR product, 1µl of cloning vector and 1µl of DNA ligase in a centrifuge tube. Carefully mix them, incubate 10 minutes at room temperature and put on the ice.
Transforming and selection of positive clones: firstly, add connection products into the 50µl of Trans-T1 competent cells, and put the cells on ice for 20-30 minutes; secondly, put the cells at 42°C for 30 seconds, then immediately placed it on the ice for 2 minutes; thirdly, add 250µl of SOC or LB medium at room temperature and cultured cells at 200rpm, 37°C for 1 hour; fourthly, coat the flash with the mixture of 500M IPTG 8µl and 20mg/ml X-gal 40µl, and placed in 37 °C incubator for 30 minutes; after IPTG and X-gal were absorbed, the 200µl bacteria solution was evenly coated on the plate and cultured overnight in the incubator at 37°C; finally, the white clones were selected and inoculated in 50ml LB liquid medium, respectively.
