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recipes

SDS-Page Solutions

Tris-Glycine Running Buffer (10x) PAGE Gels

    • 250 mM Tris-HCl, pH 8.6
    • 1.92 M Glycine
    • 1.0% SDS (Sodium Dodecyl Sulfate)
    • Dilute to 1000 mL with DI water
Note
  • SDS is a respiratory irritant, so minimize exposure when handling using gloves, goggles, and mask.

Sample (Loading) Buffer

in 10 ml:

    • 4 ml 100% glycerol
    • 2.4 ml 1M Tris/HCl pH 6.8
    • 0.8 g SDS
    • 4 mg bromophenol blue
    • 0.5 ml beta-mercaptoethanol
    • 3.1 ml H2O

aloquot out to 200uL/1.5mL Eppendorf tube and store at -20 degrees C

Staining Solution

    • 0.25 g Coomassie Blue R-250
    • 175 mL ethanol
    • 275 mL DI water
    • 25 mL glacial acetic acid

Low-Toxicity Destain

To 500 mL of water add:

    • 400 mL ethanol
    • 100 mL glacial acetic acid


(makes 1 L total solution)

NativePAGE Solutions

Running Buffer

The final concentration of the 1X buffer is given below:
50 mM BisTris
50 mM Tricine
pH 6.8
1. To prepare 1000 mL of NativePAGETM Running Buffer (20X), dissolve the following reagents in 700 mL ultrapure water:
BisTris 209.2 g
Tricine 179.2 g
2. Mix well and adjust the volume to 1000 mL with ultrapure water.
3. Store at room temperature. The buffer is stable for 6 months when stored at room temperature.
4. For electrophoresis, dilute this buffer to 1X with water (page 13).

When made used 1/4 BisTris and Tricine in 500mL water to make a 10X solution.

Growth Media

LB Medium

For 500 mL:

  • 5 g tryptone
  • 2.5 g yeast extract
  • 5 g NaCl
  • Add 475 mL water, dissolve solids, add 0.1 mL 5 M NaOH, make to 500 mL with water. Autoclave and store at room temperature. Refrigerate after opening.
Notes
  • For agar plates, add 15 g agar prior to autoclaving
  • For ampicillin plates, add 1 mL sterile 50 mg/mL ampicillin after cooling to 50°C

TB Overexpression Medium

For 1000 mL:

  • Nutrient solution
    • 12 g tryptone
    • 24 g yeast extract
    • 8 mL 50% glycerol
    • Make to 900 mL and autoclave
  • Buffer solution
    • 2.31 g KH2PO4
    • 12.54 g K2HPO4
    • Make to 100 mL and autoclave.
  • Cool to 55 °C or less, combine, and add any antibiotics desired.
Notes
  • Store and autoclave nutrient and buffer solutions separately
  • Combine only immediately prior to usage

Other Solutions

Resuspension Buffer-DesD

For lysing cells
Final Concentrations:

    • 20 mM Tris-HCl
    • 200 mM NaCl
    • 10 mM imidazole
    • 10% glycerol
    • pH 8.0
    • 5 mM TCEP

For 1000mL

    • 20 mL 1M Tris-HCl
    • 11.685 g NaCl
    • 0.6808 g imidazole
    • 100 mL glycerol
    • 1.43325 g TCEP
    • Bring to 800mL volume with DI water and adjust pH with HCl at that time, then bring to 1 L
Notes
  • The addition of 10% glycerol was made on June 1, 2012 after significant protein degradation in both DesC and DesD purification's was observed. FPLC runs prior to this date did not include any glycerol.
  • Binding Buffer is an alternate name for resuspension buffer.
  • In August, 2016, ITC experiments showed that DesD was not maximally active without 5 mM TCEP or other reducing agent.

Elution Buffer-DesD

For FPLC
Final Concentrations:

    • 20 mM Tris-Cl pH 8.0
    • 10% Glycerol
    • 200 mM NaCl
    • 1 M Imidazole
    • 5 mM TCEP

Final Buffer(Desalting Buffer)-DesD

Final Preparation:

    • 20 mM Tris-HCl, pH 8.0
    • 200 mM NaCl
    • 10% glycerol (For sizing column)
    • 5 mM TCEP


Sizing Column Buffer

Recipe:

    • 20 mM Tris-HCl, pH 8.0
    • 200 mM NaCl
    • 1 mm TCEP
    • 5% glycerol

For 1 Liter:

    • 20 mL of 1 M Tris pH 8.0
    • 11.685 g NaCl
    • 50 mL glycerol
    • 3.145 mL of 0.318 M TCEP
Notes

-5% glycerol reduces the pressure within the column, to not load higher concentrations of glycerol. If protein solubility becomes an issue, add more glycerol to the protein once fractions elute
-TCEP is a reducing agent necessary for DesC to elute in its monomeric state.

IPTG


For 0.5 mM IPTG: 0.119 g/L
For a 500 mM stock, combine 0.119 g/mL and aloquot out in mL stocks. Store frozen at -20C

Sugars and Antibiotics

TE Buffer

For preparation of oligo stocks (storage at -80)
Final concentration:

    • 10mM Tris Cl pH 8.0
    • 1mM EDTA

Equivalents for 100mL:

    • 1mL of 1M Tris Cl pH 8.0
    • 200uL of 0.5M EDTA

TAE 50x stock solution

    • 242 g Tris base (FW = 121.14)dissolved in approximately 750 mL deionized water.
    • 57.1 ml glacial acid acid
    • 100 mL of 0.5 M EDTA (pH 8.0)


adjust the solution to a final volume of 1 L, without further adjustment of pH.
This stock solution can be stored at room temperature.
A dilution to 1x is appropriate for DNA gels.

ITC Buffer


DesD binding and kinetics buffer (do not use Tris)

    • 100mM Hepes pH 7.5
    • 200mM NaCl
    • 5mM TCEP
    • 25% Glycerol (final)
    • 5mM MgCl2 (can increase to 10mM)


FslA binding and kinetics buffer (do not use Tris)

    • 100mM Ches pH 9.0
    • 200mM NaCl
    • 5mM TCEP
    • 25% Glycerol (final)


For desalting in the FPLC, a maximum of 10% glycerol should be used to avoid straining the pumps. Dialyzing overnight into a higher %gly will have a concentrating effect on your sample, though, so dialyze into 25% buffer.

FslA Buffer Recipes

Grams provided are for a 1L stock solution

Resuspension Buffer (Buffer A)

  • 50mM Ches-HCl pH 9 (10.36g)
  • 200mM NaCl (11.688g)
  • 10mM imidazole (0.6808g)
  • 5mM TCEP (1.433g)
  • glycerol (10-15%) (100mL-150mL)

Elution Buffer (Buffer B)

  • 50mM Ches-HCl pH 9 (10.36g)
  • 200mM NaCl (11.688g)
  • 500mM imidazole (34.04g)
  • 5mM TCEP (1.433g)
  • glycerol (10-15%) (100mL-150mL)

Desalting Buffer (Final Buffer)

  • 20mM Ches-HCl pH 9 (4.1458g)
  • 200mM NaCl (11.688g)
  • 5mM TCEP (1.433g)
  • glycerol (10-15%) (100mL-150mL)