Koch Lab:Protocols/Microsphere-DNA tethering/Glass, dig, biotin, microsphere, 4kb DNA

Materials

Sample chamber

You can use more sophisticated sample chambers, obviously, but our most common method remains the ultra-low-tech "double-stick tape" method. For this you need:

• #1 coverglass (or whatever your microscopy requires). 60 x 24 mm is convenient. You can get Corning from Fisher.
• regular glass slides (or you can use another coverglass). 1 inch x 3 inch, about 1.2 mm thick.
• 3M double-stick tape (like the kind you can get at Office Max).

Anti-dig

Polyclonal sheep anti-dig from Roche (Cat. No. 1 333 089). This is shipped as a lyophilized powder. We always resuspend entire 200 microgram bottle with 1 ml of ice-cold PBS, and then make 20 microliter aliquots which are stored at -80C. An aliquot can be extracted from freezer, and diluted with 180 microliters of cold PBS to make:

20 microgram / milliliter working solution of anti-dig
keep cold, store at +4C for up to a few weeks, or until you run out, or until stuff stops working

Procedure

Form tethers

This assumes you have a typical sample chamber made out of #1 coverglass, double stick tape, and microscope slide (or another coverglass). Assumes sample volume about 10-20 microliters. Assumes glass is already cleaned, and dry.

1. Coat the glass surfaces (and presumably the sticky tape walls) with anti-dig