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Knight:Protein DNA Binding/option 3
Knight:Protein DNA Binding/option 3
1 Stock solutions
o 1.1 500mM HEPES-NaOH pH 7.9
o 1.2 20mg/mL BSA
o 1.3 1M magnesium chloride
o 1.4 4M potassium chloride
o 1.5 20mM zinc sulfate
2 Making binding reaction buffer
3 Binding reaction conditions
Stock solutions
500mM HEPES-NaOH pH 7.9
To make 200mL
20mg/mL BSA
1M magnesium chloride
To make 100mL
MgCl2 is very hygroscopic. May want to order a fresh bottle since bottles should not be stored
for a long time.
4M potassium chloride
To make 100mL
Dissolve 29.82 g solid KCl in 60 mL H2O.
Adjust volume to 100 mL with H2O.
Autoclave for 20 minutes on liquid cycle and store at room temperature.
Aliquot into 100 μL aliquots in sterile tubes and use one at a time.
Note that this concentration of potassium chloride solution is near the solubility limits for
potassium chloride in water. So you may need to heat slightly to help the salt go into solution.
To make 100mL
To make 10mL
2 mL 50% glycerol
300 μL 500mM HEPES-NaOH
250 μL 4M potassium chloride
50 μL 1M magnesium chloride
50 μL 20mM zinc sulfate
50 μL 20mg/mL BSA
To make 100mL
20 mL 50% glycerol
3 mL 500mM HEPES-NaOH
2.5 mL 4M potassium chloride
500 μL 1M magnesium chloride
500 μL 20mM zinc sulfate
500 μL 20mg/mL BSA
Add β-mercaptoethanol just before use (0.7 μL per mL of buffer).
Or add TCEP to final concentration of 1mM (2μL of 0.5M stock per mL of buffer).