Validated CALR Lentiviral shRNA #V81146
Calreticulin; Sicca Syndrome Antigen A (Autoantigen Ro; Calreticulin); Endoplasmic Reticulum Resident Protein 60; Calregulin; CC1qR; CRP55; ERp60; HACBP; Grp60
NCBI Gene Entry: 811
Human CALR mRNA knockdown
Product should be stored at –80°C
Note: The following protocol uses HeLa cell as an example assuming that your cell culture medium is DMEM-based. If you use a different cell culture medium, please contact us for a special quote or technical support.
- Release 0.5 million HeLa cells into a 35 mm tissue culture dish in 2 mL of the growth medium (DMEM containing 10% FBS and 1% pen/strep). Cell density should reach 50-60% confluence the following day.
- 24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
- Discard 1 mL of the original growth medium of the 35 mm dish.
- Using a serological pipette, gently mix the lentiviral solution 3 times.
- Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
- Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
- 48 h after cell release, without discarding the original medium, add another 1mL of lentiviral medium directly into the dish.
- Add an additional polybrene stock solution into the dish to obtain a final concentration of 5 μg/mL. Tip: Now, the medium in the dish should be a total of 3 mL.
- 72 h after cell release, cells may reach confluence. Trypsinize the cells off the 35 mm dish and culture those cells in a 60 mm dish.
- Add puromycin to the dish at a final concentration of 4 μg/mL. Tip: To assess the efficacy of puromycin selection, culture a dish of wild-type HeLa cells as a negative control.
- Allow puromycin selection for 48 h. Almost all wild-type HeLa cells should die, while the dish infected with lentiviruses should have some remaining cells.
- Replace the medium with the regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.