Biomedical Engineering Reference
In-Depth Information
PROTOCOL 10.17 Screening ES Cell Colonies for Homologous
Recombination of Targeting Vector
Equipment and reagents
•
Water bath at 55
◦
C
•
PCR reagents and primers
•
Digestion buffer (50 m
M
Tris
-
HCl, pH 8.0; 1 m
M
CaCl
2
; 1% Tween-20 in dH
2
O; 10mg/ml
proteinase K).
Method
1 Resuspend harvested ES colony pellets (
for PCR screening
)in50
μ
lofdigestion
buffer.
2 Let digestion proceed at 55
◦
Cfor6h.
3 Denature proteinase K in each tube by immersing the tubes in boiling water for
10min.
4 Centrifuge the digestions briefly to collect evaporated droplets and allow to cool to
room temperature.
5Use2
μ
l of each digested colony for one PCR reaction.
bb
Notes
bb
The exact PCR screening strategy will depend on the targeting vector, primer design and
experimental conditions (see Figure. 10.1). Nevertheless, it is generally recommended to
perform several screens per colony, including sets of primers to test for:
1 The quality of the DNA prep, by screening an endogenous region not targeted for
modification
2 The efficiency of the primers used to screen for correct homologous recombination
3 Random insertion of the targeting construct
4 Homologous recombination of the targeting construct at the targeted locus
PROTOCOL 10.18
Expansion of Correctly Targeted ES Cells
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
•
10ml glass pipettes
