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.
|