Field of Science

Quick, change focus!

(I wrote this two days ago, but forgot to click 'Publish post'.)

The optical tweezers apparatus is now working well, so I'm off across town tomorrow to give it a try, with the expert help of my biophysicist collaborator.  But can I remember what I had learned so far, and what the status of my DNA-on-beads preps is?  No.  So I'm glad I wrote some blog posts and kept a tolerably good notebook.

I can attach biotinylated DNA to streptavidin-coated polystyrene beads.  The problem that the DNA caused the beads to clump together was resolved by agitating the beads better while they were incubating with the DNA (by putting the tubes on the roller in an orientation where they would be turned end-over-end).  However this resulted in beads that were difficult to pellet for washing, a problem that hasn't been solved but can be circumented by washing the beads by filtration and then concentrating them with a disposable protein-concentrator (Amicon).
  • I need to find the bead-DNA preps I made and take them home with me tonight.
I can get cells to adhere nicely to coverslips if I rub the coverslips with ~10 ┬Ál of poly-L-lysine solution until dry.  It's easy to then assemble the treated coverslips into 'chambers' for the tweezers work.  I mark an 'X' near the center of the coverslip (on the coated side) with a fine-point sharpie; this makes it easy to be sure I'm focusing in the right place with the tweezers apparatus.  To prepare chambers with cells in them, I add the cell suspension to the chamber, incubate it coverslip-side down for 1-2 minutes, and then rinse thoroughly with 5-10 volumes of competence medium (rapping several times to dislodge weakly bound cells) before sealing with candlewax.  The attached cells are still alive and able to grow and divide if given culture medium.
  • I will prepare some more coverslips and chambers before I go home tonight, and take them with me.
I have frozen competent cells (H. influenzae and B. subtilis) in the -80 °C freezer at the biophysics lab.
  • But I suspect there may be contamination in the fridge stock of competence medium I use to wash the chambers and to wash and resuspend the cells in after thawing (to remove the glycerol antifreeze), so I should take some fresh stock with me and leave it in the -20 °C freezer rather than in the fridge.
Tomorrow the first objectives will be to make sure I can trap cells with the laser, and I can see the cells on the coverslip.  If that works, we can try bringing a bead to a cell and see if it attaches!

No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS