CRISPR puromycin
Rebecca Berrens
Abstract
This protocol is for CRISPR knock-in generation in mouse embryonic stem cells
Before start
Have ES cells ready growing happily. Mostly 2 passages after thawing
Steps
Plate cells on 6-well plate on SNL a day before transfection (number of cells shown in the table below).
Transfect cells using Lipofectamine 2000 protocol and plasmid DNA amounts as shown in the following table:
| A | B |
|---|---|
| 6:1 Molar Ratio dTAG-SETDB1 to MA299/300 | No DNA Control |
| 100uL OPTIMEM + 6.46 uL (2.5 ug) dTAG-SETDB1 + 0.44 uL (869.3ng) MA299/300 | 100ul OPTIMEM |
| 100uL OPTIMEM + 10 uL LF2000 | 100uL OPTIMEM + 10uL LF2000 |
Transfection guidline
A day after transfection cells were passaged from 6-well plate into 10cm Petri dishes with SNL. Cells from each well were split into 3 Petri dishes in 3 different densities (1/2, 1/3 and 1/5 of the cells).
24 hours later apply puromycin selection (at the adequate concentration for your cell line of interest) for 48hours to select for cells that contain the Cas9 plasmid (there is no selection in the targeting vector!).
After 48 hours of puromycin selection feed cells with regular ES medium every day until ready to pick colonies (usually 12-14 days after transfection).
pick colonies (usually 12-14 days after transfection).
- follow ES cell picking protocol
