Aug 17, 2022
CRISPR puromycin
- 1IDRM, Paediatrics, Oxford University
Protocol Citation: Rebecca Berrens 2022. CRISPR puromycin . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmndpog3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 17, 2022
Last Modified: August 17, 2022
Protocol Integer ID: 68784
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
Plate cells on 6-well plate on SNL a day before transfection (number of cells shown in the table below).
30m
Transfect cells using Lipofectamine 2000 protocol and plasmid DNA amounts as shown in the following table:
| E14 1x10^6 cells | E14 1x10^6 cells | |
| 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).
30m
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!).
10m
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).
10m
pick colonies (usually 12-14 days after transfection).
- follow ES cell picking protocol