Team:NAU-CHINA/Protocols
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Protocols
Transient transfection of Jurkat T cells
1. Culturing of cells before nucleofection. Cells should be nucleofected after reaching 70-85% confluency, higher cell densities may cause lower nucleofection efficiencies, mycoplasma contamination will negatively influence experiments results.
2. Combine the cells of interest, DNA and the appropriate cell-type specific Nucleofection Solution (Here Buffer C), and transfer to an amaxa certified cuvette (invitrogen cuvette). 1X106-5-106 cells, 1-5 μg plasmid DNA in 1-5 μL H2O or TE, and 100 μL room temperature Buffer are used in this step. The quality and the concentration of DNA used for nucleofection plays a central role for the efficiency of gene transfer. We strongly recommend the use of high quality products for plasmid purification like Qiagen endofree plasmid kit. The purified DNA should be resuspended in deionised water or TE buffer with a concentration between 1-5 μg/μL. The ration of A260;A280 should be at least 1.8 for nucleofection.
3. Insert the cuvette into the Nucleofector TM and choose the cell-type specific program. Press the start button "X". If the program is unknown, please following the recommend above. To avoid damage to the cells remove the sample from the cuvette immediately after the program has finished (display showing "OK").
4. Rinse the cuvette with culture medium and transfer the cells into the culture dish.
5. Incubate at 37 °C until use.
| Chemical | Stock Conc. | Final Conc. | 40 mL | 200 mL |
|---|---|---|---|---|
| Buffer B | ||||
| NaCl | 5 M | 137 mM | 1.1 mL | 5.5 mL | KCl | 1 M | 5 mM | 0.2 mL | 1 mL |
| Na2HPO4 | 0.5 M | 0.7 mM | 56 μL | 280 μL |
| Glucose | 1 M | 6 mM | 240 μL | 1.2 mL |
| HEPES(pH7.0) | 0.1 M | 20 mM | 8 mL | 40 mL |
| dH2O | 30.4 mL | 152 mL |
| Chemical | Stock Conc. | Final Conc. | 40 mL | 200 mL |
|---|---|---|---|---|
| Buffer C | ||||
| KCl | 1 M | 120 mM | 4.8 mL | 24 mL | CaCl2 | 1 M | 0.15 mM | 6 μL | 30 μL |
| Na2HPO4 | 0.5 M | 10 mM | 0.8 mL | 4 mL |
| Glucose | 1 M | 6 mM | 240 μL | 1.2 mL |
| HEPES(pH7.6) | 1 M | 25 mM | 1 mL | 5 mL |
| EGTA | 0.1 M | 2 mM | 0.8 mL | 4 mL |
| MgCl2 | 1 M | 5 mM | 0.2 mL | 1 mL |
| dH2O | 32.1 mL | 160.8 mL |
Lipofectamine® 2000 DNA Transfection Reagent Protocol
1、Seed cells to be 70–90% confluent at transfection.
2、Dilute four amounts of Lipofectamine® Reagent in Opti-MEM® Medium Opti-MEM® Medium.
3、Dilute DNA in Opti-MEM® Medium.
4、Add diluted DNA to diluted Lipofectamine® 2000 Reagent (1:1 ratio).
5、Incubate for 20 minutes at room temperature.
5、Add DNA-lipid complex to cells.
6、Incubate cells for 1–3 days at 37 °C. Then analyze transfected cells.
| Culture Vessel | Surface Area per well (cm2) | Opti-MEM Media | DNA (μg) | Lipo 2000 (μL) |
|---|---|---|---|---|
| 12-well | 4 | 50 μL *2*12 | 1.0 | 3.75 |
| 6-well | 10 | 100 μL*2*6 | 2.0 | 7.5 |
| 60-mm | 20 | 500 μL *2*1 | 4.0 | 24 |
| 10-cm | 60 | 1 mL*2*1 | 8.0 | 40 |
Calcium Phosphate Transfection
| 2X HBS | For 1 pter |
|---|---|
| HEPES | 10 g |
| KCI | 0.74 g | Glucose | 2.4 g |
| NaCl | 16.3 g |
| Na2PO4 | 0.214 g |
Dissolve, in 950 mL H2O. Adjust pH to exactly 7.5 with 1 M HCL, bring up to 1 L with ddH2O. Filter using 0.22 micron filter. Aliquot in 50 mL Falcon tubes. Store at -20 ℃.
2 M CaCl2: Filters sterilize. Store in 1 mL aliquots at -20 ℃. Stable at RT.
Transfection
1. Twenty-tour hours prior to transfection, inoculate 1-2×106 cells/10 cm plate in 10 mL DMEM/F12 media + 10% BCS supplemented with L-glutamine and Pen/Strep. At the time of transfection, cells should be about 60-70% confluent.
2. The following day (20-24 hours later), transfect cells. Up to 15 to 20 μg DNA can be used for one 10-cm plate. If more than one plasmid is used for transfecting the same plate, equalize DNA amounts among different plates by adding vector plasmid DNA to some plates, so that the total amount of plasmids used for each plate is the same. For transfection.
a. Prepare the following mix for 10 cm plate:
61 μL 2 M CaCl2
10 μg DNA total
Add ddH2O to 500 μL
Then vortex to mix.
b. Slowly (drop-wise) add 0.5 mL of 2x HBS while typing the mixture prepared in (a).
c. Drop total 1 mL of the final mix around the plate, then mix gently. Place the plate back in the incubator.
3. Twelve to 16 hours after transfection, gently aspirate the medium and add 10 mL of pre-warmed fresh medium to the plates. Try not to disturb the fine DNA-CaPO4 precipitates on the bottom of the plate.
4. The following day, harvest the cells and extract protein as needed.
RT-qPCR
Extraction of RNA
1. Suck and discard DMEM culture solution. Wash cells with 500 μL PBS, suck and discard PBS. Add 300 μL trypsin to digest the cells. Add the same amount of 10 % DMEM culture solution to stop the digestion. Transfer the cell suspension to a 1.5 mL EP tube and centrifuged at 1000 rpm/min for 5 min using a centrifuge. Suck and discard the supernatant , and add 600 μL PBS to resuspend the cells. Add 500 μL trizol to 170 μL cell suspension, mix well, transfer the mixture to 1.5 mL