Continue to grow 96-well plates (feed every day) until the plates are confluent for isolation of genomic DNA. If you are not using the 96-well feeder plates for a mini-Southern, you may use them as a back up for expansion, freeze down, or isolating DNA.
Allow colonies to grow in 24-well plates (continue to feed every day). Let the plates reach ~80% confluence. If you have already analyzed your potential clones just expand and freeze down your positive clones. You can split your positive clones from a 24 to a 12-well plate and freeze 2 vials/well to have a backup of all your good clones. Freeze cells down in 12-well plates using standard freezing ES cell protocol. If expanding for genomic Southern and a freeze; split the 24-well plate 1:2 1/2 for freeze 1/2 for genomic DNA.
1. Feed plates with NO DRUGS 2 hours before freezing.
2. Prepare 12-well plates with feeders and ES cell media
3. Prepare freezing media 20% DMSO, 80% FBS, and label appropriate # of 1.2 ml freezing vials
4. Aspirate off ES cell media and wash with PBS
5. Add 100 ul/well of trypsin (0.05%) and incubate for 5 min. at 37 C
6. Add 150 ul/well of FBS. Titrate and scraping gently the bottom of the dish.
7. Add 125 ul to each well of a 12-well plate and 125 ul to each cryovial.
8. Place all 12-well plates in incubator -rock to spread cells when placing inside incubator.
9. Add 125 ul of freezing media to all vials serially.
10.Place in -80 C; transfer to liquid N2 within the week.
Forms Services Links Return to Protocol Main Page Return to ES Core Homepage