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!

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1) CUT 4 PIECES OF 3MM WHATMAN PAPER AND PLACE EACH ONE IN A SEPARATE CONTAINER(A CAFETERIA TRAY WILL PROBABLY WORK PERFECTLY).

2) SATURATE EACH OF THE 4 PIECES WITH ONE OF THE FOLLOWING SOLUTIONS:
A) 10% SDS
B) Denaturing Solution (.5N NaOH, 1.5M NaCl)
C) Neutralizing solution (1.5M NaCl,.5M Tris pH 7.4)
D) 2X SSC

** DO NOT ALLOW THE FILTERS TO BECOME TOO WET BECAUSE THE COLONIES WILL SWELL AND DIFFUSE DURING LYSIS.

3) PLACE THE NITROCELLULOSE FILTERS COLONY SIDE UP ON THE SDS SATURATED PAPER. ALLOW TO INCUBATE 3 MINUTES.

** YOU MAY WANT TO TRANSFER THE FILTERS TO DRY WHATMAN PAPER BETWEEN TREATMENTS.

4) TRANSFER TO DENATURATION SATURATED PAPER FOR 5 MINUTES.

5) TRANSFER TO NEUTRALIZATION SATURATED PAPER FOR 5 MINUTES.

6) TRANSFER TO 2X SSC SATURATED PAPER FOR 5 MINUTES.

7) ALLOW TO DRY AT RT 30 MINUTES. CROSSLINK.

PREWASH

1) FLOAT BAKED FILTERS ON 2XSSC, ONCE COMPLETELY WET SUBMERGE 5 MINUTES .

2) TRANSFER TO PREWASH SOLUTION
5X SSC
.5% SDS
1mM EDTA PH 8

3) GENTLY SCRAPE BACTERIAL DEBRIS FROM SURFACE OF FILTERS USING KIMWIPES SOAKED IN PREWASH SOLUTION. INCUBATE AT 50 DEGREES FOR 30 MINUTES.

4) TRANSFER TO PREHYB SOLUTION.