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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!

DEPHOSPHORYLATION OF LINEARIZED DNA
ALKALINE PHOSPATASE(CIP) DIGESTION

Digestion of protruding 5'-ends
1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer.
2. Add 1U CIP for 100pmol 5'-ends.
2g linearized 5kb plasmid Å 1.4pmol 5'-ends
3. Incubate 30' @ 37oC.
4. Add 1ml 0.5M EDTA (to 5mM).
5. Phenol-Sevag extract to remove all the enzyme and NaOAc/EtOH ppt.
6. Resuspend in ddH2O or TE-4 @ Å100ng/l.

Calf Intestinal Alkaline Phosphatase
Boehringer Mannhein #713 023

0.1M Tris-HCl, pH 8.5
10mM ZnCl2
10mM MgCl2

Digestion of blunt or recessed termini
1. Precipitate digest DNA and resuspend in 100ml 1X CIP Buffer.
2. Add 1U CIP per 2 pmol -ends.
3. Incubate 15' @ 37oC.
4. Add another aliquot of CIP and incubate an additional 45' @ 55oC.
5. Stop rxn by adding 1ml 0.5M EDTA, phenol-Sevag extract and EtOH ppt.

Dephosphorylation of RNA
To remove 5' phosphates from RNA, use 0.01U CIP/pmol 5' termini. Incubate 15'@ 37oC, followed by 30' @ 55oC.
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