User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2010/10/22

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Tubulin suspension

 * The main constituent of microtubules is the tubulin, which is a globular protein. The tublin is stored in Tubulin Storage Buffer (TSB).

Tubulin Storage Buffer
The recipe for TSB is:

1858 µl of 1.06xPEM

20 µl of 100 mM GTP

120 µl of Glycerol

2 µl of MgCl2


 * Preparation of 1.06xPEM

We will prepare solution with a total volume of 2000 µl. Therefore, we will have to dilute 180 µl of 10xPEM in 2810 µl of distilled water.


 * Preparation of the suspended GTP

To prepare the 100 mM solution of GTP, get 191.4 µl of the 1.06xPEM and pour it into the 10mg jar of GTP. Take the inner container of the jar and seal it with paraffin. Then, gently tap it against the counter to get the chemical to go down from the walls.

Finally, transfer the whole volume to a 0.5 ml microcentrifuge tube.


 * Obtaining the accurate amount of Glycerol

Glycerol is a very thick liquid, so to pipette it is a difficult task. First, submerge the tip of the pipette as little as possible and let it stay in that position until the liquid stops going up the tip.


 * Final step in the TSB preparation

In a cryogenic vial, pour the needed amounts of 1.06xPEM, 100 mM GTP, and MgCl2. Then, insert the tip of the pipette with Glycerol and start “wetting” the tip until all the Glycerol is gone. Finally, centrifuge the TSB and make 2 ml aliquots.

Tubulin Suspension
In the lab we have two types of tubulin: Un-labeled bovine tubulin and rhodamine-labeled bovine tubulin. We prepare suspensions of each type in TSB.

Before preparing the suspensions, get a bucket with liquid Nitrogen. BE REALLY CAREFUL WITH IT!


 * Un-labeled bovine tubulin suspension.

The un-labeled bovine tubulin comes in small containers with 1mg of tubulin and they are covered with a white cap. The concentration is 5 mg/ml of tublin in TSB.


 * Procedure

Add 200 µl of TSB to the vial with the tubulin. Draw the solution in and out with the pipette to make sure it is mixed properly. Once ready, keep the vial in the electronic ice bucket. Then, prepare 5 µl aliquots in small microcentrifuge tubes. As soon as an aliquot is ready, it needs to be placed in the bucket with liquid Nitrogen. After preparing all the aliquots, carefully remove them from the bucket with liquid Nitrogen and place them in a bigger tube. Label the big tube, and store it in the -80oC freezer.


 * Rhodamine-labeled bovine tubulin suspension

The rhodamine-labeled bovine tubulin comes in small containers with 20 µg of tubulin and they are covered with a red cap. The concentration is 5 mg/ml of tubulin in TSB. The rhodamine fluorophore give the characteristic red color to this tubulin.


 * Procedure

Add 4 µl of TSB to the vial with the tubulin. Draw the solution in and out with the pipette to make sure it is mixed properly. Once ready, keep the vial in the electronic ice bucket. Then, prepare 2 µl aliquots in small microcentrifuge tubes. As soon as an aliquot is ready, it needs to be placed in the bucket with liquid Nitrogen. After preparing all the aliquots, carefully remove them from the bucket with liquid Nitrogen and place them in a bigger tube. Label the big tube, and store it in the -80oC freezer.

29% Rhodamine-labeled tubulin (Combination of un-labeled and Rhodamine-labeled tubulin)
In a separate vial, put the contents of one of the 5 μl aliquots of the un-labeled tubulin suspension and the contents of one of the 2 μl aliquots of the Rhodamine-labeled tubulin suspension. Then, prepare 1 μl aliquots (discard the last one: the 7th one). Store them in the -80oC

Get one of the 1 μl aliquots and put it on the thermocycler for 30 min. Ten minutes before the 30 minutes are over start the preparation of Taxol.


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