User:Linh N Le/Notebook/2009/08/26: Difference between revisions
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==To Do== | ==To Do== | ||
* Re-suspension of tubulin rehash | * Re-suspension of tubulin rehash | ||
* Make GPEM | |||
==Tubulin== | ==Tubulin== | ||
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*I will try to find all the notes from the wiki from the 3 notebooks of the 3 members above | *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 | *Note: there are instructions that come with the concentrated tubulin | ||
** [[User:Andy Maloney|Andy Maloney]] 00:58, 27 August 2009 (EDT): Do you know where these are? | |||
** [[User:Steven J. Koch|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? | |||
** [[User:Linh N Le|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 [http://www.openwetware.org/wiki/User:Linh_N_Le/Notebook/2009/06/15 6/15/2009] | *My notes say that we resuspended the tubulin on [http://www.openwetware.org/wiki/User:Linh_N_Le/Notebook/2009/06/15 6/15/2009] | ||
*[http://openwetware.org/wiki/User:Steven_J._Koch/Notebook/Kochlab/2009/06/15/TRITC_%28Rhodamine%29_and_regular_tubulin Koch's notes] here as well | *[http://openwetware.org/wiki/User:Steven_J._Koch/Notebook/Kochlab/2009/06/15/TRITC_%28Rhodamine%29_and_regular_tubulin Koch's notes] here as well | ||
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***Ideally the LN2 level should be such that the liquid is splashing the top of the racks | ***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 | *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=== | ===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''' | |||
===Unlabeled Tubulin=== | ===Unlabeled Tubulin=== | ||
*Same as above but we made 38, 5ul aliquots in the 500ul tubes | *Same as above but we made 38, 5ul aliquots in the 500ul tubes | ||
*We added 200ul 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 | *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 | *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 | |||
*'''Final Concentration 5mg/ml | |||
==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 [http://www.openwetware.org/wiki/User:Anthony_Salvagno/Notebook/Research/2009/05/12/Microtubule_creation 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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Revision as of 14:26, 27 August 2009
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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
Unlabeled Tubulin
GPEM
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