User:Linh N Le/Notebook/2009/08/26: Difference between revisions
From OpenWetWare
Line 18: | Line 18: | ||
*Koch also have [http://openwetware.org/index.php?title=Koch_Lab:Protocols/Kinesin/Tubulin_resuspension_and_polymerization&oldid=307098#Non-labeled_tubulin_resuspension a great page] about resuspending tubulin and polymerizing it | *Koch also have [http://openwetware.org/index.php?title=Koch_Lab:Protocols/Kinesin/Tubulin_resuspension_and_polymerization&oldid=307098#Non-labeled_tubulin_resuspension a great page] about resuspending tubulin and polymerizing it | ||
==Procedure== | ==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 is[[Koch Lab:Protocols/GPEM|GPEM]] | *The first thing we need is[[Koch Lab:Protocols/GPEM|GPEM]] | ||
*Last time I made 300 microliters total (294 ul [[Koch Lab:Protocols/PEM|PEM]] + 6 ul [[Koch Lab:Protocols/GTP|GTP]]) | *Last time I made 300 microliters total (294 ul [[Koch Lab:Protocols/PEM|PEM]] + 6 ul [[Koch Lab:Protocols/GTP|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 | |||
===Rhodamine Tubulin=== | |||
*When we resuspended the Rhodamine lableled (also called TRITC Tubulin) we made 4, 1ul aliquots | |||
*Add 4 ul of ''Cold'' GPEM+cushion to the stock tube of TRITC Tubulin 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 | |||
*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 | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
|} | |} |
Revision as of 13:34, 26 August 2009
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
To Do
Tubulin
ProcedureNote: It is important to keep the tubulin cold, so prepare all the tubes, buffers and LN2 before you start
Rhodamine Tubulin
|