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Team:University of Chicago/Notebook/Buffers
From 2008.igem.org
Contents |
Buffers
Enzyme Buffers
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
- Note: Create 1M concentration stocks to make buffer prep easier in future.
CCMB80: with aqueous stocks
- 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.
