User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/15/Making Tubulin

GPEM

 * 294 uL PEM
 * 6 uL GTP

For a total of 300 uL of GPEM

GPEM + Cushion

 * 9 uL GPEM
 * 1 uL Cushion

Rhodamine Tubulin
The GPEM + Cushion was chilled. From here we took one aliquot of rhodamine tubulin from the -80 freezer. We then added 4 uL of the GPEM + Cushion. We very quickly mixed the solution and pipetted 4 uL of of the resuspended tubulin into 4 aliquots (each containing 1 uL of TRITC). These four aliquots were then flash frozen in liquid nitrogen.

Some of the tubulin had stuck to the side of the aliquot (rather than the bottom), so after flash freezing we spun them until all of the TRITC was at the bottom of the aliquot. We again flash froze the aliquots and then placed 3 in the -80 freezer (we kept one out so that we could see if it worked).

Unlabeled Tubulin

 * 180 uL cold GPEM
 * 20 uL cushion
 * 1 mg unlabeled tubulin

We used 180 uL of cold GPEM and added this to one mg of tubulin (one aliquot). We then addded 20 uL of cushion and mixed. From here it was a race against room temperature to get get the tubulin into aliquots. I pipetted 5 uL of tubulin into aliquots, which were then handed to Lihn and promptly placed in a bath of liquid nitrogen. We were able to make 38 aliquots which were put into larger tubes and placed in the -80 C freezer.