Transient Transfection
From CCGB
Transient Transfection Assay (Dual Luciferase) using Invitrogen’s Lipofectamine LTX with PLUS Reagent
-- by Christine Dorman
Description The goal is to transfect K562 cells with our predicted cis-regulatory(pCRM) modules fast, accurately, and efficiently. The pCRM’s are cloned into a Firefly(FF) luciferase reporter vector and Renilla(Ren) luciferase is used a cotransfection control. K562 Cells are transfected using Lipofectamine LTX with PLUS Reagent. Cells are grown for 48hrs post transfection then harvested and tested for FF and Ren. Luciferase activity.
Reagents and Supplies
• Rapidly growing K562 cells split into antibiotic free media 24hrs before transfection below passage number 20
• DMEM Dulbecco’s Modified Eagle Medium Invitrogen#12430 supplemented with 10% calf serum (Refrigerator in TC room)
• OPTI-MEM Reduced serum medium Invitrogen #31985 (Refrigerator in TC room)
• Lipofectamine LTX Reagent Invitrogen #15338-100 (Refrigerator in TC room)
• Plus Reagent Invitrogen #11514-015 (Refrigerator in TC room)
• Sterile microcentrifuge tubes and tips
• Sterile 24 well tissue culture plates
• Plasmid DNA to transfect 4 wells of a 24 well plate for each construct tested (if DNA is to be compared must all be prepped from the same kit and quantitate via a spectrophotometer)
• Promega’s Dual Luciferase Assay Kit
Procedure for Transfection
1. Collect and count K562 cells. Plate cells at a density of 1.8x105 cells/well/ml. Plate 1ml of cells in media without antibiotics/well. Place in 370C incubator.
2. Label 24 well plates and tubes to be used for your transfection.
3. Put the calculated amount of OptiMEM in each tube.
4. Put the calculated amount of Renilla Luciferase in each tube.
5. Put your test Construct in the appropriate tube.
6. Gently vortex Plus Reagent. Add Plus Reagent to each tube. Vortex lightly let stand for 5 min. at room temp.
7. Gently vortex Lipofectamine LTX and add appropriate amount to each tube. Vortex lightly and let tubes stand for 30 min. at room temp.
8. After incubation add 100μl of the DNA-Lipofectamine complex to each well. Mix gently.
9. Place plates in incubator and let grow for 48hrs.
Note: When setting up a transfection always set up 4 wells for every pCRM and calculate an extra .75 wells to make sure there is enough Lipofectamine-DNA complex for all 4 wells.
Procedure for Harvest and Assay
1. Remove plates from CO2 incubator. Bring to main lab.
2. Remove cells and media from each well into pre-labeled 1.5ml microtubes
3. Spin down for 1 min. 10000 rpm in eppendorf centrifuge.
4. Pour off supernatant then vacuum remaining media off of cell pellet
5. Resuspend cell pellet in 100 μl of 1X passive Lysis buffer. Let stand at room temp for at least 15 minutes.
6. Turn on luminometer and go thru the set-up procedure so it will have time to warm up
7. Prepare Luciferase Assay Reagent II (LARII). May be some aliquoted, may have to make fresh. Make sure if you thaw aliquots you pool the aliquots to the amount that you need to ensure the reagent is uniform.
8. Pipet 50 μl of LAR II into glass tubes for the luminometer
9. Prepare appropriate amount of Stop& Glo Reagent, 50 μl/tube. Stop & Glo comes as a 50X stock and the kit supplies the buffer to dilute the reagent.
10. Vortex the lysed cells, remove 10 μl and pipet into 50 μl of LARII, mix by shaking the tube gently, place in luminometer and take a reading.
11. Remove tube, add 50 μl of prepared Stop&Glo, vortex and take another reading.
12. Remove tube from luminometer and discard.
13. Continue with remaining samples.
Transient Transfection Assay (Dual Luciferase) using Tfx-50 Lipofectant
-- by Hanna Petrokowska
Night before the experiment:
Prepare Tfx-50 (Promega # E1811, order through Fisher) by adding 400ul of nuclease-free water (provided) to a vial of dryed Transfectant. Vortex for 10 sec, hold under 60oC water bath for 60 s, vortex again and store at –20o C before use.
For K562 cells use 0.8ug of total DNA, a Tfx-50:DNA ratio of 2:1 in a 2ml final volume, and 4x105 cells per transfection, in triplicate. (To obtain this ratio, multiply the ug DNA by 3 to get the correct volume of Tfx-50 to add. It will be 2.4ul in this case).
As a internal control use 0.008ug of Renilla plasmid (in addition to 0.8ug of your firefly construct DNA).
1. Warm up plain DMEM Medium (serum free) in 37C water bath.
2. Count cell and wash 2x enough for n+1 samples in sterile 1x PBS. Suspend pellet in pre-warmed serum-free medium at 1x107 cells/ml (1x106 cells/0.1ml medium).Will use 40ul of cell/well.
3. During the washing of the cells prepare Tfx-50/DNA mixture:
a) label plates b) add 0.4ml serum free, pre-warmed Medium to each well c) add 0.8ug of your DNA to each well d) make Tfx-50/Renilla mix (2.4ul/0.008ug for each well) e) add to the medium in the well and mix for 10 minutes, but no more than 15 minutes.
4. Aliquot 4x105cells (40ul @ 1x107cells/ml) into each well and allow to incubate for 2 hours in 5% CO2, 37oC incubator.
5. After 2 hrs add 1.6ml (2ml total) complete medium (10% BCS, 2% PSF in DMEM ). If you are performing Hemin induction use 40uM of Hemin in the Medium.
6. Harvest cell cutures after 40 to 48hrs for Dual Luciferase Assay. (Centrifuge cell in the microfuge at 2000rpm for 5 minutes and wash 2x with non sterile 1xPBS buffer. Completely remove supernatant by aspiration using a 200 ul pipet tip attached to aspirator hose. Make n+2 volumes of lysis buffer, Dilute 5X Dual luciferase passive Buffer to 1X with water. Suspend pellet in 100 ul lysing buffer and incubate at RT for 10 min. Centrifuge at full speed for 1 min in Eppendorf microfuge. Use a toothpick to remove pellet. Use 20ul of the cell lysate for Luciferase Assay or store frozen at –80oC until use.)
Transient Transfection Assay (Dual Luciferase) using Tfx-50 Lipofectant
-- by Hao Wang
In a 5 ml petri dish, use 1×106 cells, 2μg of DNA and 6μl of Tfx50 (1:2). If using 12 well plate, change amount of DNA, cell and Tfx50 proportionally, that is 4×105cells, 0.8μg of DNA and 2.4μl of Tfx50 for a total volume of 2ml.
We used to add 0.005μg-0.01μg of Renilla construct in addition to 2μg of firefly construct. Proportionally, 0.004μg-0.008μg of Renilla will be added for a smaller volume.
Prepare mixture of plasmid, Tfx50 and plain medium.
Take a plasmid with concentration of 0.5μg/μl as an example.
First, make a 1:10 dilution so the concentration is 0.05μg/μl. Dilute Renilla to final concentration of 0.001μg/μl Plain medium needed: (400-2.4-0.8/0.05-8)×3.2=1195.52μl=1.196 ml Tfx50 needed: 2.4×3.2=7.68μl Firefly plasmid needed: 0.08/0.05×3.2=51.2μl Renilla plasmid needed: 8×3.2=25.6μl
This mixture will be sufficient for 1 triplicate.
It’s easy to make an Excel file to handle multiple samples.
1. Mix plain medium +DNA (FF+Ren) +Tfx50, count 10 min.
2. During this 10 min, count cells and adjust density to 1X107cells/ml.
3. Aliquot 40μl of cells into each well.
4. Add 0.4ml of mixture into each well.
5. incubate 1-2 hour(s), warm complete medium.
6. Add 1.6ml of complete medium into each well, continue to incubate for no more than 48 hours.
