Team:University of Chicago/Notebook/Buffers

Enzyme Buffers
[[Media:Protein Buffers.pdf|A good collection of buffers used in enzymology.]]

CCMB80

 * 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
 * 80 mM CaCl2.2H2O (11.8 g/L)
 * 20 mM MnCl2.4H2O (4.0 g/L)
 * 10 mM MgCl2.6H2O (2.0 g/L)
 * 10% glycerol (100 ml/L)
 * adjust pH DOWN to 6.4 with 0.1N HCl if necessary
 * adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
 * sterile filter and store at 4°C
 * slight dark precipitate appears not to affect its function

CCMB80: with aqueous stocks
 * Note: Create 1M concentration stocks to make buffer prep easier in future.
 * 10 mM KOAc pH 7.0: 10mL of 1M stock/L
 * 80 mM CaCl2.2H2O: 80mL/L
 * 20 mM MnCl2.4H2O: 20mL/L
 * 10 mM MgCl2.6H2O: 10mL/L
 * 10% glycerol: 100 ml/L

Hutner's Mineral Base Concentrate
It is not difficult, but follow directions carefully.


 * 20g nitrilotriacetic acid (NTA)
 * 54.5g magnesium sulfate heptahydrate
 * 6.67g calcium chloride dihydrate
 * 18.5mg ammonium molybdate tetrahydrate
 * 198mg iron sulfate monohydrate (or 323.7mg of the heptahydrate)
 * 100mL Metals-44

TO BE CONTINUED

5x Ligation Adjustment Buffer

 * Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
 * KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)
 * CaCl2 400 mM (200 ml/l of a 2 M solution)
 * MnCl2 100 mM (100 ml/l of a 1 M solution)
 * Glycerol 46.8% (468 ml/liter)
 * pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)
 * Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST)
 * water to 1 liter
 * autoclave or sterile filter
 * Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
 * Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.