Andy is back! He wants to try the kinesin again, maybe his expertise will allow us to succeed
Make BRB80
12-5
Tubulin
Stumbled upon a site that details how they get tubulin from cow's brains
BRB80
Since Andy and Koch are the only people in the lab that know how to make a buffer (I actually dont know if Ant can), we waited til Andy came back to try to make BRB80
Not to mention the fact that we are also running out of the stuff we got from Cytoskeleton (new shipment will be in this week)
It is a "universal" buffer whose ph changes linearly with the amount of alkali that is added to it
It consists of:
.04M H3BO3 (Boric Acid)
.04M H3PO4 (Phosphoric Acid)
.04M CH3COOH (Acetic Acid)
The stuff we use is also known as PEM
0.1 M PIPES (pH 6.95)
Piperazinediethanesulfonic acid C8H18N2O6S2
2 mM EGTA
ethylene glycol tetraacetic acid C14H24N2O10
1 mM MgSO4
Magnesium Sulfate
Also can make it as
100 mM Pipes,pH 6.9
1 mM MgCl2 (magnesium chloride)
1 mM EGTA
SJK 01:58, 23 June 2009 (EDT)
Ph Titrations
Recalling what little i remember from freshman chemistry, Andy has instructed me to create 40uL of a "normal" base of NaoH
Normal meaning 1M soln
So, we look up the molar mass of NaOH
MM 39.9971 g/mol
If I want a 1 mol soln, i need 39.9971 g in 1L
Means I need 3.9971g in a dL
Means I need .39971g in a ml
Means I need .039971g in a ul
Meaning I need 1.598g in a 40ul soln
Thats wrong, new train of thought
Lets try to make .4L of 1M soln
I need 15.99884 g for .4L of water (4 deca liters)
divide by 10, 1.599884g for a 40ml
Making the Buffer
We measured out the following
1mM EGTA (0.076g)
80mM PIPES (4.83g)
1mM MgCl2 (.2mL)
For a 200mL stock of the Buffer (which Andy hates naming, but I will name KLB)
We titrated it with NaOH to raise the Ph to 6.89
Used about 26ml of NaOH to get there
Added an entry to the Kochlab inventory page, under the VWR fridge category (Steve Koch 02:07, 23 June 2009 (EDT): Thank you! Excellent notes, as always. I really appreciate it.)