User:Anthony Salvagno/Notebook/Research/2009/05/12/Microtubule creation

Reference protocol

http://openwetware.org/index.php?title=Koch_Lab:Protocols/Kinesin/Tubulin_resuspension_and_polymerization&oldid=307098

Resuspending ATP and GTP

Steve Koch 13:18, 12 May 2009 (EDT): Anthony adding 100 microliters of cold DI water (from CHTM supply) to both of the lyophilized tubes from cytoskeleton. This gives us 100 mM of both ATP and GTP. Need to look up what the buffer is (i.e. does it have Mg).

• Steve Koch 13:49, 12 May 2009 (EDT): Made my first couple mistakes: First, didn't notice that ATP was supposed to be resuspended in 1 milliliter of 100 mM Tris-HCl pH 7.5. Also, called cytoskeleton and the woman asked someone who said the GTP is sodium GTP. Thus, we wanted to resuspend in 100 mM MgCl2, not water (even though the tube said to resuspend in water.)

Correcting mistakes in ATP, GTP

Steve Koch 14:46, 12 May 2009 (EDT): Anthony was able to get 1 M Tris-HCl, pH 7.5 and 1M MgCl2 from Kelly (thanks, Kelly!).

• Had previously added 100 ul of water to ATP. Was supposed to add 1 ml of 100 mM Tris pH 7.5, so, need to add:
  • 800 ul of cold water
  • 100 ul of 1 M Tris.
  • Done
• Have 100 mM GTP (sodium) in water (or buffer?). Have 1M MgCl2. To make it equimolar of MgCl2.
  • 5 ul MgCl2
  • 50 ul resuspended GTP in water
  • 45 ul of cold H2O

Making GPEM

• 4 ul of 50 mM GTP-MgCl2
• 196 ul of PEM (from cytoskeleton)
• Technically, this is slightly less than 1x PEM, Final concentration of 1 mM GTP

Making GPEM + cushion

• 90 ul of GPEM
• 10 ul of cushion buffer from cytoskeleton (this pipetted easily with the 10 microliter eppendorf pipetman)

FITC Tubulin, aliquots

1. Combined all the 5 tubes of FITC tubulin into one tube. Even after slight losses, there seemed to be 10 micoliters in the combined tube. Added 10 microliters of cold GPEM+cushion. Made 2 microliter aliquots which were flash frozen, and stored at -80C in thick-walled 500 microliter tubes. Tried to seal them with parafilm, but didn't do a good job.
   • Starting concentration was 10 mg / ml w/o glycerol. http://www.cytoskeleton.com/products/tubulins/t332m.html
   • After adding 10 microliters of GPEM + cushion, was 5 mg / ml, the appropriate concentration for polymerization.
2. put one 2 microliter aliquot in thermal cycler for 22 minutes.
3. stabilized them with 198 microliters of warm BRB80-T
   1. To make BRB80-T
   2. Resuspended one taxol aliquot with 100 microliters of room temperature DMSO. This makes 2 mM stock.
   3. Added 5 microliters of 2 mM stock DMSO to 995 microliters of PEM (from cytoskeleton)

Success!

Steve Koch 19:44, 12 May 2009 (EDT): We made a simple sample just by putting 5 microliters of stabilized MTs onto a slide and dropping a coverglass on top of it. We did not dilute into antifade solution or anything. I think we used the 60x oil immersion high NA objective, but not sure. We made a sharpie mark on the inside of the coverglass to make it easy to focus. Using our existing scope and FITC cube. It was easy to see LOTs of nicely polymerized MTs. They faded pretty quickly (no antifade), but it was easy to see that the polymerization was highly successful.

Setting up autoclave

Steve Koch 14:39, 12 May 2009 (EDT): Tuttnauer Brinkman model 3545.

• Leveling with 400 ml of water. Just need to adjust the front down a bit. Actually it's close enough.
  • Dumped water out afterwards
• Filling with DI water to the line inside the storage tank.
• Followed instructions, will post those here later. Now running a dry cycle.