Due to popular request, I’m going to put my *most current* version of the HEK 293T Landing Pad recombination protocol here for others’ benefit. Much of the credit goes to Sarah Roelle, who wrote up this current version of the protocol.

Recombination:

[This protocol is for a 24-well plate:]
Day 1:

1) Make 2 transfection mixtures per sample:
Tube 1: 23 μL Opti-MEM + 1 μL Fugene6

Tube 2 (If using cells that don’t already express the Bxb1 recombinase enzyme): μL of DNA corresponding to 16 ng Bxb1 plasmid + μL corresponding to 224 ng attB plasmid and OPTI-MEM to a final tube volume of 24 μL.
-OR-
Tube 2 (If using cells that already express the Bxb1 recombinase enzyme): μL of DNA corresponding to 240 ng attB plasmid and OPTI-MEM to a final tube volume of 24 μL.

2) Mix Tube 2 into Tube 1 for each sample. Mix up and down a couple times with pipet. Then let the mixtures sit for 15-30 min while you get the cells ready (Unless you trypsinized and counted the cells first, to know how many you had in case they were limiting).

4) [Meanwhile] Trypsinize and count the cells. Add 120,000 cells per well in a final volume of 300 μL media to each well.

5) Once at least 15 minutes have passed since mixing, add the mixtures dropwise throughout the well of cells being transfected.

Day 2:
Add at least 500 μL media to each well.

Notes about scaling up / down:
We typically pilot new plasmids in a 24-well scale, and transfect larger populations of cells by transfecting 6-wells and increasing the number of plates / wells as needed.

Negative selection (if applicable):

Negative selection with iCasp9 is wonderful, and hopefully you are using one of those landing pad versions. I typically use 10nM final concentration of AP1903, although I’m fairly certain 1nM is just as effective. Death occurs in a couple of hours, so you can come back to your plate / flask later on in the day and change media to get rid of the dying cells. I typically wait until at least 72 hours after recombination to perform this step.

Important note: If you’re doing a library-based experiment, then make sure you leave some cells aside (even 100k to 500k cells will be plenty) that DO NOT go through any selection steps, since this will allow us to estimate the number of recombined cells (and thus estimate the coverage of your library; see below for the calculation). If you’re just doing individual samples, this isn’t nearly as big of a deal, since you’ll be able to visually inspect the number of recombinants (ie. do you see only a handful of surviving cells, or is it 100+ individual cells surviving?).

Positive selection (if applicable):

I tend to do the positive selection step AFTER negative selection, since the negative selection step has usually thinned the number of cells down enough such that the positive selection is going to be most effective (I find that over-confluent wells tend not to do super-grant with positive selection). Thus, while it can be done as soon as 72 hours post recombination, I tend to do this a week or later after recombination. You can find effective concentrations for positive selection of landing pad HEK 293T cells here.

Some additional notes:

1. Due to a phenomenon of (annotated) promoter-less expression from the thousands of un-recombined plasmids that remain in the cell following transfection, I typically wait ~ 5 to 7 days before running any cells in the flow cytometer, to wait for that background signal to die down.
2. Other transfection methods may work, but will need to be optimized. For example, I have observed transfection with lipofectamine 3000 to result in prolonged promoter-less expression, probably due to increased transfection and greater toxicity preventing cell division and thus plasmid dilution.
3. Calculating the number of recombinants. OK, so as I alluded to above, it’s definitely worth estimating the number of recombinants of any library-based experiments. To do this, take your observed recombination rate from running flow on your unselected cells (say, you see 5% of cells as being mCherry+ / recombined when you ran flow on unselected cells 7 days after transfection), and multiply that fraction with the number of cells you transfected. So, if we pretend that you transfected 20 million HEK 293T cells and you had observed 5% of cells recombined in your unselected well, then the rough calculation is 2e7 cells * 0.05, amounting to roughly 1 million cells estimated to have been recombined. Of course, directly sequencing what’s there in the recombined library is a more direct measurement of library coverage at the recombined cell step, but doing this calculation is still usually worthwhile as another line of evidence.