EXO/S1 DELETION SERIES

 1. Cut DNA with 5'- and 3'- overhangs, gel check, phenol-Sevag extract and NaOAc/EtOH ppt.

2. Prepare one S1 nuclease tube (on ice) for each time point: 15ml S1 Buffer + 0.25U S1/ml (5U).

3. Use 5ml (=0.5mg) digested plasmid (in EB) per time point. Add 4U Exonuclease III/ml reaction mix and incubate @ 37℃ (‰400 digested/min).

4. Remove 5ml aliquots into the S1 nuclease tubes (on ice) at various times. When all time points have been collected, incubate the tubes 30' @ RT.

5. Add 2ml STOP Buffer/tube and incubate 10' @ 70oC.

6. Add: 2.0ml 10X Klenow Mix

+2.0ml 0.125mM dNTPs

+0.5ml Klenow

Incubate 10' @ 37℃.

7. Analyze 10ml on a gel. To the remainder in each tube, add 40ml Ligation Mix + 1ml T4 DNA Ligase. Incubate overnight @ 15℃ (or 2-3h @ RT).

8. Transform 100ml competent cells with 10ml ligation reaction; freeze remainder.

EB 66mM Tris-HCl, pH 8 0.66mM MgCl2 10X Klenow Mix 20mM Tris-HCl, pH 8100mM MgCl2 S1B 40mM KOAc 300mM NaCl 1.3mM ZnS04 7% Glycerol Ligation Mix 50mM Tris-HCl, pH7.61mM MgCl2 1mM ATP1mM DTT5% PEG 6000 S1 STOP 300mM Trizma base50mM EDTA