User:Anthony Salvagno/Notebook/Research/2009/08/06/DNA Unzipping

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Yesterday Koch made some fork tethers (to be drawn up later). The problem is, according to him, the tethers are only 60-80bp long from single stranded floppy end to double stranded unzipping side. This is a problem because with our uncalibrated and noisy tweezers it is difficult to determine what exactly we are doing to the tethers. We have some promising results, but we would like some definitive answers. I feel like that last sentence was straight out of one of those history channel science documentaries.

Unzipping Tethers

I will make 4 tethering samples:

  1. dsDNA 4.4kb for stretching from here on out denoted as dssDNA (with the second s meaning stretching)
  2. 3 unzipping constructs:
    • library DNA for shits and giggles
    • TD cause its uber long
    • tube 3 cause it could work the best (that tube and the other 3 need to be relabled)

Tethering

Ok so I made aliquots of 1:10 and 1:100 of TD(1), Tlib(2), and T(3). I made the 1:100's first and then decided 1:10 would be better. There is 50uL of the 1:10 (in each tube) and 100uL of the 1:100.

The naming convention goes:

  • T means tetherable
  • next series of letters/numbers for the original stock going way back
  • (_) for which tube (they are numbered). IE there are 2 TD tubes 1 and 2, so TD(1) means tube 1.

Tethering:

  • Note: I am also doing one no DNA.
  • Following normal protocol.
  1. Flow anti-dig and wait 5 min
  2. flow 5sv BGB and wait 2 min
  3. flow DNA and wait 5 min
    • so far so good, no overflows or anything... hope i didn't just jinx myself.
  4. flow pop
  5. flow beads wait 7 min
    • I ran out of regular beads after flowing in Tlib, T(3), and TD
    • for 4.4dssDNA I used the remained of Koch's salt+beads
    • the no DNA chamber has about 5uL of high concentration beads. I just pipetted out of stock cause I was in a pinch.
  6. flow pop
  7. seal chambers

Analysis

I found that there were very few tethers in the 4.4dssDNA and maybe about the same in the T(3) chamber. Although in that one it was also really hard to find stuck beads, so who knows. No DNA had nothing but stuck beads, and Tlib had nothing as well. TD had one tether right away but the bead fused to the surface and we couldn't find anything else.