Dear Mark,

You effectively need to follow how fast your cells are growing not to have overpopulated plates at the time of transfection. You might have to consider to check the efficiency of the knockdown between 24 and 48hour after transfection.

You might be better not to plant the cells and transfect the same day. Althought your cells might be robust, they might be a bit disturbed by planting and transfecting the same day.

Best

Gilles

Dear Mark,

I agree with Gilles, depending on the sturdiness of the cells plating and transfecting the same day might not be ideal for cell survival. Why don't you plate a dilution series of cells and use the wells that gave you 60-80% the next day? Also, I would always test such conditions for each cell line I use. 

Best,

Meike

Dear mark,

Transfection efficiency might be reduced if your cells are over confluents. And their survival will be reduced if they are less confluent thus you might not enough cells as much as you would like to have to run you experiment.

However, Jurkat cells are non-adherent so I do not think they would need recovery time after splitting as much as adherent cells need. So if you insist on merging day 0 and day 1, make sure that you reach the 60-80% confluence at transfection.

I also recommend performing the transfection after like 5-6 hours after splitting the cells.

Good luck,

Rakan.

Dear Mark,

I also think you should wait for having 60-80% of confluence. If you have problem with Lipofectamine, I advise you to have a try with Viromer BLUE, you can reach more than 60% of efficiency...

Depending on how long you want the cells to be knocked down on your target gene, you can also do two-rounds of knockdown starting with less then 50% confluence. I routinely do this way and it can keep the gene knocked down for three days.

Mark: The last part of your question is about fast-forward method and/or reverse transfection method.Owing to the nature of Jurkat cells (non-adherent cells that behaves more like suspension cells). Hence, you may have to go for traditional transfection method.