User:Steven J. Koch/Notebook/Kochlab/2009/08/27/Possible unzipping constructs
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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)
- antidig 5 minutes
- 5 s.v. BGB Pop; repeat for total of 3
- 1.5 s.v. DNA (or 1X pop); incubate 5 minutes
- 5 s.v. BGB Pop; repeat for total of 2 times
- 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
- 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
