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Protocols
	ES Cell Preparation for Microinjection

1)      Coordinate the growth of your ES cells with the transgenic injection facility (Angela will check the number of plugged blastocyst donors). You will want to thaw your ES cell clones for injection 3-4 days prior to the anticipated injection day.

2)      Split ES cells (~50% confluent) 1:5 one day prior to the planned injection day.

3)      On injection day, trypsinize the ES cells to a single cell suspension. Generating single ES cell is important for microinjection.  (mechanical disruption of ES cell clumps by pipetting through a p200 tip is often effective).

4)      Inactivate trypsin with blastocyst injection media.

5)      Centrifuge cells and resuspend in 1 ml of blastocyst injection media (small cryovials are good).

Blastocyst Injection Media

    Low glucose D-MEM             44 ml

    FCS                                            5 ml   

    500mM HEPES                          1ml

1. Combine the D-MEM, FCS, and HEPES in a 50 ml conical tube.

2. Filter through a 0.2-micron syringe filter into a sterile 50 ml conical tube.