User:Linh N Le/Notebook/2009/08/26

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(Rhodamine Tubulin)
(Rhodamine Tubulin)
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*Once all 4 are done, then remove the rack from the LN2 and send it to the -80C freezer
*Once all 4 are done, then remove the rack from the LN2 and send it to the -80C freezer
**Note: if you want to polymerize one for use that day, no need to flash freeze that tube
**Note: if you want to polymerize one for use that day, no need to flash freeze that tube
-
*'Final Concentration 5mg/mL
+
*'''Final Concentration 5mg/mL'''
===Unlabeled Tubulin===
===Unlabeled Tubulin===

Revision as of 17:26, 27 August 2009

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To Do

  • Re-suspension of tubulin rehash
  • Make GPEM

Tubulin

  • Andy needs to know how Brigette, Koch, and I resuspended the tubulin from stock to make the little aliquots that we then polymerize
  • I will try to find all the notes from the wiki from the 3 notebooks of the 3 members above
  • Note: there are instructions that come with the concentrated tubulin
    • Andy Maloney 00:58, 27 August 2009 (EDT): Do you know where these are?
    • Steve Koch 01:07, 27 August 2009 (EDT): There was talk of getting a spikey-thingy and I think Linh even used his own money to get one. If not there, could be in the plastic folder thingy Ant put on the -20C freezer. Or in the cytoskeleton folder?
    • Linh N Le 01:58, 27 August 2009 (EDT) for one, Brigette bought the spike. Secondly, there are old instructions in plastic bags hanging on the side of the small -20 freezer. Third, each "bag of aliquots" that needs resuspending comes with one. Brigette and I read the instructions located on the fridge to make some of these notes
  • My notes say that we resuspended the tubulin on 6/15/2009
  • Koch's notes here as well
  • Brigette's Notes
    • We made both Rhodamine and unlabeled tubulin
  • Koch also have a great page about resuspending tubulin and polymerizing it

Procedure

Note: It is important to keep the tubulin cold, so prepare all the tubes, buffers and LN2 before you start

  • The first thing we need isGPEM
  • Last time I made 300 microliters total (294 ul PEM + 6 ul GTP)
  • We also need to add a special cushion buffer (provided by Cytoskeleton), so Koch mixed it up with the Gpem
    • 9 μL GPEM
    • 1 μL cushion
  • Have the proper amount of 500 ul tubes ready
  • Have a ice bucket with LN2 ready to go (we used the red ice bucket/tray since it had the highest surface area)
    • In the LN2 should be the plastic tube racks (4 legged, 16 slot white or black) ready to go
      • Ideally the LN2 level should be such that the liquid is splashing the top of the racks
  • Also have an Ice bucket with ice ready so that when you take out the tubulin to mix and aliquot out you can keep it on ice
  • The manuals state that both of the tubulins must have a concentration of 10mg/ml to work

Rhodamine Tubulin

  • When we resuspended the Rhodamine lableled (also called TRITC Tubulin) we made 4, 1ul aliquots
  • The manual states to spin the original tube down to get all the tubulin to the bottom (a small red dot)
  • Add 4 ul of Cold GPEM+cushion to the stock tube of TRITC Tubulin (20 ug) and mixed it a bit using the pipette man.
  • Pipette 1ul of the solution into a tube then place the tube into the LN2 (make sure the sample is at the bottom of the tube, we had to centrifuge one down and although it didnt seem to hurt the tubulin, its always safer not to have to do this)
    • This was accomplished using 2 people, one to pipette the tubulin out and one to immediately add it to the LN2
  • We did seal the tops with parafilm, but this is a large time sink and was deemed unnecessary by Koch for the unlabeled
  • Once all 4 are done, then remove the rack from the LN2 and send it to the -80C freezer
    • Note: if you want to polymerize one for use that day, no need to flash freeze that tube
  • Final Concentration 5mg/mL

Unlabeled Tubulin

  • Same as above but we made 38, 5ul aliquots in the 500ul tubes
  • We added 200ul of cold GPEM+cushion (180ul GPEM + 20ul cushion) to a 1mg tube of the tubulin (notice the higher concentration of the unlabeled kind, hence more aliquots were made), mixed it and put 5ul into a 500ul tube, then flash froze
  • For this, we used the red icebucket and 5 racks to fit all the tubes into the LN2
  • The tubes were then placed into a 50ml falcon tube for storage in the -80C

GPEM

  • Brigette and I are going to make a stock of GPEM for use since Andy expressed a want to resuspend tubulin soon
  • Following instructions left by Ant
  • We have PEM premade from before so we will make it with that
  • Looking for GTP, notes say that there is 50ul of it in the -20c Freezer
  • Koch notes also state that said GTP is make with water and we want to make it with mgcl2
    • There is a tube of premixed MgCl2 on the wet lab shelf (1M MgCl2 made 8/21/07)
  • To correct the GTP
    • 5 ul MgCl2
    • 50 ul resuspended GTP in water (this is in the freezer)
    • 45 ul of cold H2O
  • This gives us 100ul of sodium GTP
  • Hey! Guess what? Brigette found the old GPEM and GPEM + Cushion in the e-Ice bucket near the PH Meter
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