CRISPR Transfections

Transfection of HEK 293 Cells


Day1: Plate Cells


Plate cells: For a 12-well, or 24-well, plate out 4,800,000 cells per plate (400,000 or 800,000 cells/well, in 500μl or 1ml media, respectively).



Day2: Transfection


Reagents:

Lipofectamine® 3000 Transfection Reagent

For 12-well plate use 1μg of DNA per well, according to manufactures protocol:

total DNA:	1000ng
	gRNA:		227.27ng
	Cas9:		772.73ng


For 24-well plate use 500ng of DNA per well, according to manufactures protocol:

total DNA:	500ng
	gRNA:		113.64ng
	Cas9:		386.36ng

For 24-well plate:

Tube 1:	
	OptiMem:	25µl
	L3000:		1.5µl
     
Tube 2:	
	OptiMem:	25µl
	P3000:		1µl
	DNA:		(500ng)

Mix well, incubate 5 minutes, and then add 50μl to each well.

Day3-5: Harvest Genomic DNA

Reagents:

QuickExtract Solution

Genomic DNA prep:

Harvest cells in QuickExtract Solution (Epicentre QE09050)

Dissociate cells and spin down at 300g for 5 minutes

Remove media and add QuickExtract Solution (use ~1μl per 10,000 cells and keep volume to minimum)

Vortex 30sec and spin down

Incubate at 65C for ~20 minutes with intermittent vortexing

Incubate at 95C for ~15 minutes

Vortex, spin down, store are -20C

Electroporation of Stem Cells


Coming Soon