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

Notes

1. I would call this the "Lazy protocol," or "Sufficient protocol." One could probably do better by increasing incubation times, tweaking concentrations, being careful with temperatures, being precise with incubation times, etc. But for ripping through a bunch of samples in one afternoon, it seems to work well.

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

Kim wipe wicks

We use twisted Kim wipes as wicks for drawing fluid out of one side of the cell. Steve likes to keep it folded in half, and twist from one corner of the fold.

Procedure

Create a sample chamber

Details later (it's tough to describe, but easy to learn from someone)

1. Clean glass
2. Make chambers
   • We find that making a bunch of chambers ahead of time doesn't save that much time, and they may not stay clean, and may losing sticking strength. So we make them on demand.

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
   • Because the sample is dry (and clean/hydrophilic), the liquid will flow in without the need for a wick on the opposite side. If it doesn't flow in easily, and bubbles form, it's probable that your glass isn't clean enough.
   • During this step, measure the volume of your sample chamber, which will be determined by the width of the channel. You can make a good guess by eye, dial it in on your pipetman, and then make a good estimate based on how under- or over-filled it gets. From here on out, we will call this the "sample volume" or S.V.
   • Flow in one S.V. of 20 microgram/ml antidig.
   • Let sit at room temperature for 5 to 10 minutes.