Plasmid transfection
Introduction
Materials
- Puromycin dihydrochloride (Life Technologies, cat. no. A11138-03)
- Lipofectamine 2000 transfection reagent (Life Technologies, cat. no. 11668027)
Procedure
Preparation of cells for transfection (16~24 hours before transfection)
- Plan what DNAs you would like to transfect. It is recommended to add a positive control, such as a GFP plasmid, to monitor transfection efficiency.
- Seed the cells at a density of 1.3 x 105 cells per well in a 24-well plates in a total volume of 500µL without antibiotics.
CRITICALIt is important to seed the cells at the recommended density to maximize the transfection efficiency.
On the day of transfection
- Check the cells are at 70-90% confluency.
- This protocol is designed for transfecting cells seeded in 24-well plates. You can use this table to adjust the amount of cells and reagents you need for different conditions:
A A | B B | C C | D D | |
1 1 | 96-well | 24-wll | 6-well | |
2 2 | Surface area per well (cm2) | 0.3 | 2 | 10 |
3 3 | DNA used per well | 100 ng | 500 ng | 2500 ng |
4 4 | Lipofectamine 2000 per well | 0.2-05 ul | 1-2.5 ul | 5-12.5 ul |
5 5 | Medium to add after 24 hours | 100 ul | 500 ul | 2500 ul |
- Prepare your transfection reagents:
(1) If your gRNAs are cloned into pSpCas9(BB), transfect 500ng of your plasmid.
(2) If you transfect more than one plasmids, mix them at equimolar ratios and use no more than 500ng of total DNA.
- Dilute DNA (total 500ng) in 50µL of Opti-MEM. Mix gently.
CRITICALDo not let the diluted DNA sit more than 30 minutes.
- Add 2µL Lipofectamine 2000 to 50µL of Opti-MEM. Mix it gently and incubate it at RT for 5 min.
CRITICALDo not let the diluted Lipofectamine sit more than 30 minutes.
- Combine the diluted DNA with the diluted Lipofectamine 2000 (total volume: 100µL).
- Mix it gently and incubate it for 20 min at RT.
This step is to make the DNA and Lipofectamine 2000 to form complexes. The exomplexes are stable for 6 hours at RT.
- Add the 100µL of DNA-lipid copmlexes to each well gentally. Mix it gentally by rocking the plate.
Some cells are easy to detach from the plate. It is important to gentally to add the solution in cells to ensure optimla transfection efficiency.
24 hours after transfection
- Check cells after 24 hours for transfection efficiency.
If you transfect GFP plasmids as a positive control, you can use the percentage of fluoresccent cells to approximate the transfection efficiency.
- Supplement the culture medium with an additional 500µL of warm D10 medium.
CRITICALAdd D10 medium slowly to the side of the well as the cells can detatch easiliy.
CRITICALDo not use cold medium.
- Optional: Use 1-3µg/mL puromycin to select cells with Cas9 and gRNA successfully transfected.
48-72 hours after transfection
- Harvest cells for indel analysis.
This protocol is adapted from Ran et al., Nature protocols, 2013 and the protocol from Lipofectamine 2000 from Invitrogen.