User:Linh N Le/Notebook/2009/06/19

From OpenWetWare

< User:Linh N Le | Notebook | 2009 | 06
Revision as of 21:05, 19 June 2009 by Steven J. Koch (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

To Do

  • Autoclave more tips
  • Work on Kinesin if Brigette comes in
  • Organize the wiki

Autoclave

  • Round 1
    • 1 bottle of small centrifuge tubes
    • 1 bottle of large centrifuge tubes
    • 2 boxes 1000μL tips
    • 1 box 100ul tips
    • 1 box 10ul tips
  • Round 2
    • We have no-sterilized tips for the 2.5ul pipetteman
    • 6 boxes of 2.5ul (labeled 10ul) tips
    • They are in the new eppindorf boxes with a black background

Hiring

  • I finally got hired!
  • I just need to head down to HR on Lomas and University to fill out paperwork
  • I'm doing a timesheet right now
    • I believe Koch and I came to an agreement that the month of May was a "work for free" period with a pay increase the rest of the summer to make up for it.
      • As such, i will only "report" time from Jun1-now

Kinesin

  • I polymerized a batch of the RMTs while Brigette made some kinesin
  • She aliquoted ~6 tubes of kinesin (1ul ea), flash froze and stored in the -80c
  • She is making the motillity assay we made last time, with the same casein (the BGB koch made).
  • The first go with the kinesin was a bust
    • It was different this time, as there were alot more tubes stuck to the glass than last time
  • We decided to try a few things at the same time differently
    • first, i made a new BGB using the old powder and even older brb80 that koch made 5 yrs ago
      • We used that to coat the glass
    • Second, Brigette was afraid that there wasnt enough ATP so she tripled the concentration
  • We did 2 samples, one with same kinesin + super atp
  • old kinesin + super atp + more BRBCT
    • The thinking here was that Brigette made a too high on kinesin concentration and so we diluted it some more
  • Suffice it to say, there was not very much change in what the samples looked at from earlier.
  • I did take a video of tubes bleaching and exploding!

What could have went wrong

  • Besides the obvious "we messed it up"
  • The BGB could be bad
    • doubtful because Koch has made assays with BGB before
    • Steve Koch 22:05, 19 June 2009 (EDT):I'll have to dig around, but I'm not sure I've done motility assays with BGB (it works great for DNA tethering, but not sure about motility--not out of the question that it is bad for motility)
  • Brigette mentions that Koch's recipe for kinesin was off the top of his head, so maybe koch was wrong? (Koch wrong?! Blasphemous!!!)
    • Steve Koch 22:02, 19 June 2009 (EDT): Ha! Doubting Koch is good science! In this case, I'm not sure what recipe she's talking about. The printed recipe is mostly written by people at Sandia, and based on some standard protocols (available on "the kinesin home page". There's no doubt we're cutting some corners here as we start up, and we'll keep working to take on more of those steps (such as making BRB80, which we should do next week).
  • The kinesin could be terribly slow and not see-a-ble
    • on that note: Brigette used the "leftovers" of the vial, so that could also be an issue
      • Steve Koch 22:02, 19 June 2009 (EDT): True...lots of foam=lots of surface area = more denatured protein.
        • Steve Koch 22:03, 19 June 2009 (EDT): In the cytoskeleton order (to be placed on Monday), I ordered kits for doing "bulk" or "ensemble" activity assays--measuring ATPase activity of kinesin. This will help us know whether we have active kinesin.

Incubator

  • The temp was 27.6°C when i left at 6pm
    • Steve Koch 22:04, 19 June 2009 (EDT): Thanks for noting this!
Personal tools