Validated KDM3B Lentiviral shRNA #V4231
Knockdown Validated
Aliases
Lysine Demethylase 3B; JmjC Domain-Containing Histone Demethylation Protein 2B; Jumonji Domain-Containing Protein 1B; Lysine (K)-Specific Demethylase 3B; Lysine-Specific Demethylase 3B; Jumonji Domain Containing 1B; Nuclear Protein 5Qnca; C5orf7; JMJD1B; Chromosome 5 Open Reading Frame 7; EC 1.14.11.-; EC 1.14.11; KIAA1082; JHDM2B; 5qNCA; NET22
Background
NCBI Gene Entry: 51780
Application Information
Human KDM3B mRNA knockdown
Storage Buffer
Culture medium
Storage Instruction
Product should be stored at –80°C
Recommended Dilution
1:2
Protocols
Read all instructions carefully before using this product
Instructions For Use
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.