1. Using a sterile toothpick, pick a single clone (or cells replicated
on a master plate). Smear the cells on the bottom of an eppendorf tube.
If no master plate has been made, immediately touch the tip to replicate the clone on the
2. Add 30 ml STET buffer containing lysozyme
(8% sucrose, 0.5% Trition X-100, 50 mM EDTA, 10 mM Tris-HCl pH 8.0 and 1 mg/ml
lyzozyme. Lyzozyme optional. Add lysozyme fresh.)
3. Boil for 40 seconds.
4. Cool on ice.
6. Pipet ~3 ml 5X loading buffer onto a piece of
Saran wrap. Mix 10 ml of DNA with the loading buffer
and load on 0.8-1% agarose gel.
6. Also load the plasmid without insert. Plasmids with inserts migrate slower.
7. Loading buffer may be added to the lysis buffer, thus eliminating one more
step in this quick protocol (e.g., 50 mM NaOH, 5 mM EDTA, 0.5% SDS, 7% Ficoll, 0.01%
Bromophenol blue, 30 ml per clone, 65 °C for 20 minutes).