Expression Cloning

1. Select or prepare the expression library. High abundance of the target is very important.
2. Select a cell line to visualize positive clones. Low or no background is very critical.
   • Perform a positive control: Is this cell line capable of assembling the type of protein? A receptor/ligand pair such as PRL-R/AP-PL-I serves this purpose well.
   • As a negative control, perform the visualization on untransfected and mock transfected (i.e. no cDNA or a known negative cDNA) cells.
3. Titer the unamplified library. Plate 5000 clones per plate (LBamp). Grow overnight.
4. Using a sterile cell scraper, scrape off bacteria from plates (1 plate = 1 pool of clones), resuspend colonies in 4ml LBamp. Mix well. Without further growth, isolate cDNA from 1.5ml (Miniprep). Store 1ml samples (only one per plate needed) in 20% glycerol at -85°C.
5. Transfect cells with cDNA pools. For one well of a 6-well plate seed ca. 100,000 COS cells. If the transfection efficiency is 50%, a positive pool should show 10 positive cells (just to stress that it could very well be more than one).
6. Titer the glycerol stock of the positive pool.
   • For a 95% chance, the number of individual colonies to look at is:
     2.3 x the original colony number in the pool (i.e.: 2.3 x 5000)
   • For a 99% chance, the number of individual colonies to look at is:
     4.6 x the original colony number in the pool (i.e.: 4.6 x 5000 = 23,000)
7. These 23,000 colonies will be plated at a lower density than in the first round, e.g. 23 plates with 1000 colonies per plate.
8. Scrape, store, miniprep, transfect, visualize. Now you should see 5 x more positive cells in the positive pool (enrichment by reducing the number of colonies per pool: from 5000 to 1000 colonies per plate).
9. Go back to the stock of the positive pool, titer, plate at lower density (this time need to see 4600 colonies for 99% chance, i.e. 10 plates with 460 colonies or 20 plates with 230 colonies.)
10. Scrape, store, miniprep, transfect, visualize. Should see more enrichment.
11. Repeat working with pools of clones until it is more efficient to pick individual colonies onto a grid. Pool columns and rows by picking and combining individual colonies in a 4ml LBamp culture. Grow up in shaker, miniprep. Visualize by transfection and binding assay. (e.g., if the pool from row F is positive and the pool from column 5 is positive, the positive clone should be F5)

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