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Gene Function Analysis Using the Chicken B-Cell Line 2075. Electroporate the cells using 25 µF and 700 V (the Gene Pulser I, II, or Xcell fromBio-Rad is recommended).6. Transfer the electroporated cells to a 50-mL tube containing 15 mL CM.7. Mix well and transfer 5 mL to second 50-mL tube containing 5 mL CM.8. Aliquot the two dilutions to two labeled 96-well flat bottom microtiter plates(100 µL/well).9. Grow overnight at 41°C.10. Following 12–24 h of incubation time, add 100 µL CM containing the appropriateselective drug (blasticidin [60 µg/mL] or mycophenolic acid [2 µg/mL] or puromycin[2 µg/mL]) to each well to ensure that only cells carrying the resistance markerwill grow. End concentration is half that of the stock solution; 30 µg/mL, 1 µg/mL,and 1 µg/mL, respectively.11. Continue growing until colonies appear (normally 7–14 d). Note: some mutantsmay grow considerably slower and colonies may not appear until after 2 wk (seeNote 6).12. As soon as colonies are visible, pick single colonies by carefully pipeting 10 µL toa new 96-well flat bottom microtiter plate containing 300 µL CM/well. Continuepicking colonies daily until all single colonies possible have been picked. The bestway to see the colonies is to hold the plate up to the light and look at the bottom forwells with only single colonies. The majority of wells should have none or singlecolonies. To pick the colony, stick the pipet tip into the “center” of the colony andwithdraw the 10 µL to be transferred to the new plate. In order to assure the pickingof a single colony, one may need to perform subcloning through limited dilution onthe confirmed positive clones chosen for further use.13. Grow the picked colonies at 41°C and split and refeed CM as necessary (every 3–4 d)(see Notes 7–9).3.2.2. Targeting ScreeningTo confirm homologous targeting, the grown single colonies are checkedthrough PCR.Crude DNA Extract:1. Mix the cells in the picked colony 96-well flat bottom microtiter plate and transfer200 µL to a 96-well flat bottom microtiter PCR plate. Centrifuge for 5 min at1500 rpm and 4°C.2. Discard the supernatant and wash with 200 µL 1X PBS. Centrifuge again for 5min at 1500 rpm and 4°C.3. Discard the supernatant and resolve the pellet in 10 µL K buffer. Prepare the Kbuffer just before use.4. Spin down cells with a quick spin.5. Incubate 45 min at 56°C followed by 10 min at 95°C.6. Store at 4°C. The crude extract is stable for at least 1 wk.