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Synaptic Proteins: Protocols

Welcome to the Home of Vesicle Trafficking

A small repository of synaptic protein information


Caution! In converting these protocols to HTML, some units have been scrambled.
Specifically, microliters (ul) often appear as milliliters (ml). Think first!


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 @ 37oC (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' @ 37oC.

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

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

66mM Tris-HCl, pH 8
0.66mM MgCl2

10X Klenow Mix
20mM Tris-HCl, pH 8
100mM MgCl2

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

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

S1 STOP 300mM Trizma base

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