User:Steven J. Koch/Notebook/Kochlab/2009/08/27/Possible unzipping constructs

From OpenWetWare

Jump to: navigation, search

Steve Koch 12:37, 27 August 2009 (EDT): Want to send off a couple possible unzipping constructs:

  • short forks
    • annealed
    • individual components
  • Ant's constructs from shotgun clones
    • He showed me several tubes the other day; will have to guess at concentration

When I did the short forks a few weeks ago, they did not work for some reason. I want to try it again today to see if I can at least get tethers. Look at the tethers pre-wash to make sure flow was unzipping them

Trying fork tethers

  • Top is no DNA (6 ul sample)
  • Bottom is 10 nM fork DNA (diluted above) (10 ul sample)
  1. antidig 5 minutes
  2. 5 s.v. BGB Pop; repeat for total of 3
  3. 1.5 s.v. DNA (or 1X pop); incubate 5 minutes
  4. 5 s.v. BGB Pop; repeat for total of 2 times
  5. 1.5 s.v beads
    • Beads are marked "beads" from Ant. I diluted these to make 300 mM NaCl version.
    • 3 ul 3.8 M NaCl
    • 15 ul "beads"
    • 12 ul BGB Pop
  6. Observing before washing

Not really any more tethers in DNA sample versus no DNA (before and after washing)...possibly a few more.

Second bead try, same sample

  • 50 ul total volume
  • 10 ul beads
  • 5 ul 3.8 M NaCl
  • 35 ul BGB

Sonicated the above beads and then flowed in 1 s.v. to the first sample

Steve Koch 13:47, 27 August 2009 (EDT): still not very many stuck/jiggling beads, even at this high salt concentration

Personal tools