EXO/S1 DELETION SERIES

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 8
100mM MgCl₂

S1B
40mM KOAc
300mM NaCl
1.3mM ZnS0₄
7% Glycerol


Ligation Mix
50mM Tris-HCl, pH7.6
1mM MgCl₂
1mM ATP
1mM DTT
5% PEG 6000

S1 STOP 300mM Trizma base
50mM EDTA