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

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
 * 6) * Beads are marked "beads" from Ant. I diluted these to make 300 mM NaCl version.
 * 7) * 3 ul 3.8 M NaCl
 * 8) * 15 ul "beads"
 * 9) * 12 ul BGB Pop
 * 10) 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
Sonicated the above beads and then flowed in 1 s.v. to the first sample
 * 50 ul total volume
 * 10 ul beads
 * 5 ul 3.8 M NaCl
 * 35 ul BGB

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