Team:University of Chicago/Notebook/Buffers

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(New page: === Buffers === ==== 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 (10...)
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Revision as of 18:40, 29 July 2008

Contents

Buffers

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.
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