Validated DNMT3A Lentiviral shRNA #V2531

DNA Methyltransferase 3 Alpha; DNA (Cytosine-5-)-Methyltransferase 3 Alpha; DNA (Cytosine-5)-Methyltransferase 3A; DNA MTase HsaIIIA; EC 2.1.1.37; M.HsaIIIA; DNA Cytosine Methyltransferase 3A2; DNA Methyltransferase HsaIIIA; DNMT3A2; Dnmt3a; TBRS

NCBI Gene Entry: 1788

Human DNMT3A mRNA knockdown

The 2 mL volume is used for a 35 mm dish or a 6-well cell culture

The 6 mL volume is used for a 60 mm dish cell culture

Considering the fact that puromycin enrichment may take up to 1-2 weeks, it is strongly recommended to perform your experiment in a 60 mm dish (using the 6 mL volume) to save time. 

Culture medium

Product should be stored at –80°C

1:2

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.

1. 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.
2. 24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
3. Discard 1 mL of the original growth medium of the 35 mm dish.
4. Using a serological pipette, gently mix the lentiviral solution 3 times.
5. Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
6. Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
7. 48 h after cell release, without discarding the original medium, add another 1mL of lentiviral medium directly into the dish.
8. 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.
9. 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.
10. 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.
11. 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.
12. Replace the medium with the regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.