Genetically Engineered Mouse Core
Transgenic Mouse Core
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ES Cell Preparation Protocol

Preparation of cells for injection:

1.Feed the 6 cm plate 3 hours before using.
2.Wash with HBSS-Ca-Mg.
3.Repeat wash.
4.Add 1 ml of 0.25% trypsin to plate.
5.Incubate at 37°C for 5 minutes.
6.After trypsinization is complete, add 2 ml ES media.
7.Transfer the suspension to a 15 ml tube.
8.Using a transfer pipette, pipette up/down 20 times.
9.Centrifuge at 1000 RPM for 5 minutes.
10.Remove supernatant.
11.Resuspend cells in 3 ml of ES media.
12.Aliquot 1 ml to 1.5 ml tube for microinjection. Place this tube on ice.
13.Centrifuge the remaining 2 ml at 1000 RPM for 5 minutes.
14.Resuspend the cell suspension in 2 ml of 1 X freezing media.
15.Aliquot into freezing tubes and freeze for later use.
16.Take the ES cells that are on ice to the GEM Core by 9 a.m. the morning of scheduled injection.


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