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ES Cell Targeting

The CMGM provides in house made C and D embryonic stem cells for electroporation. Investigators who have a targeting construct ready should contact the GTTF at gttf@uchc.edu and complete an Embryonic Stem Cell Request Form. An electroporation will be scheduled upon receiving the construct DNA and a completed request form.

The CMGM will do electroporation, pick colonies to 96 well plates and duplicate the plates. The investigator will receive duplicated 96 well plates for DNA extraction and screening of targeted ES cell colonies. The positive colonies will be expanded from the master plates for aggregation with mouse embryos.

100 ug of linearized construct DNA is needed. Please use Endofree plasmid kit or CsCl-Ethidium Bromide gradients method to prepare your plasmid DNA and perform phenol-chloroform extraction after restriction digestion to linearize the construct. Please resuspend the linearized DNA in a concentration of 1 ug/ul in sterile TE or 10 mM Tris. You can also submit circular DNA.

You can ship your DNA sample by FedEx at ambient temperature by adding 1/10 volume of NaAc 3M pH5.2 and 2 volume of EtOH 100 percent into your sample. Please seal the 1.5 ml tube well and put it into a 15 or 50 ml tube. Please use air bubble envelope. The tube could be crushed easily during the shipment.

We provide at least 200 colonies for screening.

Timeline for Embryonic Stem cell electroporation:

Request Submitted Electroporation Pick Colonies 96 well-plates ready for screening     Thaw 96 well-plates,
expand ES cells,
schedule aggregation
               
               
1 to 2 weeks - expand stem cells, prepare feeders 10 to 11 days -
selection
6 to 8 days -
duplicate 96 well-plates
  Waiting for genotyping results