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Star Republic: Guide for Biologists

Screening for insertion --- plasmid size

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 master plate.

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.

5. Centrifuge.

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).