Making New ES Lines

<< previous | next >>

Initial culture conditions

Matings are set-up on Sunday and plugs are checked on Monday for delivery of mice on Thursday. On the Wednesday prior to dissecting embryos, prepare 4-well plates with PMEFs as follows:

Establishing monolayers of PMEFs


PMEF-medium/50 ml:

(1) Rapidly thaw 1 vial of PMEF-CF cells in a waterbath and transfer to 10ml of PMEF-medium. Spin down and resuspend into 25ml PMEF-medium.

(2) Transfer 0.5ml/well onto several gelatinized 4-well plates. {Depending on the expected yield of embryos, you will want 6-10 plates total}

(3) Incubate overnight. Verify the presence of a mono-layer prior to use. {Mono-layers are good for about 2 weeks}

Preparing PMEF plates for embryo culture

Prior to culturing embryos, replace PMEF-medium with SR-medium.

Recipe for SR-medium


SR-medium/50 ml:

(4) Aspirate PMEF-medium from 4-well plates and replace with 1.0ml SR-medium. Return to incubator and allow to equilibrate for several hours prior to culturing embryos. {Do this prior to fetching your uteri}


<< previous | next >>