Cellss

ES cell medium
DMEM 4500mg/ml glucose, no pyruvate, no L-glutamine
50 ml (10% ) Hyclone FCS (lot tested, special ES stock)
50 ml (10% ) newborn calf serum (lot tested, special ES stock)
6 ml 1X Penn/Strep
6 ml 1x L-glutamine (add fresh every 2 weeks)
6 ml 1x non-essential amino acids
6 ml of 7μl Sigma β mercaptoethanol in 10ml Hanks Balanced salt solution120 μl LIF-containing culture supernatent

Thawing ES cells
1. Thaw rapidly at 37˚C.
2. Transfer to 5 ml media.
3. Spin, 1000 rpm, 5 min. RT.
4. Resuspend in 10 ml media, plate on gelatin-coated plates.
(0.1% gelatin in PBS on tissue culture plates, 15-20min.)

Split ES cells
1. Wash plate with Ca/Mg-free PBS 2x.
2. Add 1ml 1x trypsin, 37˚C, 10min.
3. Pipette cells up and downthrough a 1ml pipette attached to a 200μl pipette tip. Check to ensure that the cells are single cells.
4. Pellet as above. Plate 2x106/90mm plate. Confluent at 2x107.
5. Passage every 2-3 days. Chnage media as it gets yellow.

Freezes
1. Trypsinize cells as above.
2. Resuspend cells at 5-10x106 /ml in complete medium.
3. Add 100μl DMSO/0.9 ml of cells.
4. Freeze cells at -20˚C, 2hr.
5.Transfer to -70˚C, overnight.
6. Store in liquid nitrogen.

Electroporation
1. Wash cells twice in normal PBS.
2. Resuspend 2x107 cells in 0.8ml high glucose DMEM at RT.
Straight DMEM, no penn/strep, no serum.
3. Add 10μg linearized template mixed with 20μg salmon sperm DNA to the cells.
Transfer into BioRad 0.4mm cuvette (cat#165-2088)
Stand at RT 5min.
4. Electroporate in BioRadGene Pulser. Single pulse.
250 Volts(0.25constant volts), 500μF, extended capacitance at 100 Ohms, time constant =10.0
5. Let stand in cuvette for 5 min. after electroporation.
6. Plate each cuvette into 3 90mm plates.
Transfer content into 15 ml tube.
Rinse cuvette with 2 ml ESgrow medium. Pool.
Plate 1ml into 90 mm plates containing 9 ml ESgrow.

G418 selection forNeomycin-resistant colonies.
1. After 1 day in ESgrow medium, transfer into ESgrowG418 medium.
ESgrowG418= normal ES medium containing 250μg/ml active concentration of G418.
2. Grow plate until individual well defined colonies are visible.
Mark colonies on the underside with pen.

Picking colonies.
1. Rinse plate with 1ml 1x trypsin-EDTA.
2. Repeat.
3. Scrape colony with sterile P200pipette tip and 100μl trypsin.
4. Transfer into sterile eppendorf tube with more trypsin.
Pipette up and down to homogenize.
5. Spin at 2000rpm, 2min in a microfuge.
6. Resuspend in 1ml medium. Pipette again.
7. Plate in 1 24 well dish with 2ml medium total. =Passage #2.
Grow until confluent.

First Freeze and make replica plate.
1. Rinse well with 1ml trypsin.
2. Trypsinize in1ml trypsin. Disperse with P200 pipette tip.
3. Transfer to sterile eppendorf tube.
Spin 2000rpm, 2min.
4. Suck off 900μl supernatent.
Add 1ml ESgrowG418. Resuspend with P200 pipette tip.
5. Plate 100 μl (1/10th of sample) into a 6 well dish = Passage #3.
6. Spin 2000 rpm, 2min.
7. Resuspend in 500 μl ESgrowG418.
Add 50 μl DMSO. Mix rapidly. Place on ice.
-20˚C, 2 hr.-70˚C, o/n.
Store in liquid nitrogen.

DNA isolation.
1. When the well in 6 well dish is confluent, wash 2x with 1ml Ca/Mg-free PBS
2. Trypsinize with 1 ml trypsin, 37˚C, 5 min.
3. Transfer to Eppendorf.
4. Spin 2000 rpm, 2 min.
5. Discard supernatant. Resuspend in 1 ml PBS. Store at -80˚C.
6. Use ABI DNA extractor to isolate genomic DNA.
7. Resuspend DNA in 300 μl TE.
8. Cut 40μlof DNA with restriction enzyme. Run gel. Southern blot. Probe for transgene.

Expand identified clones.
1. From Southern identify clones that carry the transgene.
2. Thaw only freeze of clone and trasnfer to 5 ml ESgrowG418.
3. Spin 1000 rpm, 5 min.
4. Resuspend in 1 ml ESgrowG418,
plate in 1 well of a 6 well plate. = Passage #4
5. When confluent, trypsinize thoroughly. Resuspend into...