ON-TARGETplus Human TET1 siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 1 × 10^5 cells per well.	Incubate transfection mixture for 10-15 min at room temperature. Replace medium after five hours and incubate for additional 48 h in in 0.2% hypoxic conditions.

Upstream tips
Seed 1 × 10^5 cells per well.
Protocol tips
Incubate transfection mixture for 10-15 min at room temperature. Replace medium after five hours and incubate for additional 48 h in in 0.2% hypoxic conditions.

Publication protocol

Cultured A172 cells were plated in six‐well plates (1 × 105 cells per well), 1 day before the transfection so that they are around 80% confluent before transfection. Next day, the cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Briefly, in different sterile microcentrifuge tubes, 100 nM siRNA against Tet1 (On‐target plus TET1 siRNA smart pool ID 80312, Dharmacon Inc, Colorado, United States, http://dharmacon.gelifesciences.com), Tet3 (Ambion, Invitrogen; ID for siTet3: 47240), or Control siRNA (On‐target plus non‐targeting siRNA, Dharmacon) were diluted in 1× opti‐MEM (Gibco). Lipofectamine 3000 (2 µl) was added to each and the mixture was immediately vortexed. This was incubated at room temperature for 10–15 minutes and media were added to the tubes to make up the volume till 1 ml. During this time the plates were washed twice with Dulbecco's phosphate‐buffered saline to remove the cell debris. After 15 minutes, the Lipofectamine 3000/siRNA/Opti‐MEM mix was vortexed and gently added over the plated cells. The plate was incubated at 37°C for 5 hours. After 5 hour, the media were changed and cells were further incubated for 48 hours in 0.2% hypoxic conditions and harvested for RNA and lysate preparation subsequently.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing siRNA / miRNA gene silencing Human - A172 TET1 using ON-TARGETplus Human TET1 siRNA from Dharmacon.

Manufacturer protocol

Download the product protocol from Dharmacon for ON-TARGETplus Human TET1 siRNA below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / miRNA gene silencing Human - A172 TET1 using ON-TARGETplus Human TET1 siRNA from Dharmacon. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.