Screening for insertion --- plasmid size

1. Using a sterile toothpick, pick single clonies and plate on a master plate. Grow overnight at 37 C.

2. Using a sterile toothpick, scrape cells from the master plate. Smear the cells on the bottom of an eppendorf tube containing 30 ml of STE (8% sucrose, 0.5% Trition X-100, 50 mM EDTA).

3. Add 30 ml phenol:chloroform:isoamyl alchol (25:24:1).

4. Vortex.

5. Centrifuge.

6. Pipet ~3 ml 5X loading buffer onto a piece of Saran wrap. Mix 10 ml of the aqueous (top) phase with the loading buffer and load on 0.8-1% agarose gel.

6. Also load the plasmid without insert. Plasmids with inserts migrate slower.