OLB
MATERIALS
Reagents
β-mercaptoethanol
dATP, dCTP, dGTP (each 100 µM in dNTP buffer)
Alternatively, use commercially available (e.g., Roche Applied Science) 100 µM dNTP stocks.
dNTP buffer (optional, as above)
2 M HEPES (pH 6.6, adjusted with 4 M NaOH)
90 OD units/mL hexadeoxyribonucleotides, in 1 mM Tris-Cl (pH 7.5)/1 mM EDTA
MgCl2
Tris-Cl
METHOD
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1. Prepare a solution containing 1.25 M Tris-Cl (pH 8.0) and 0.125 M MgCl2.
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2. To 1 mL of the Tris-Mg solution from Step 1, add 18 µL of β-mercaptoethanol and 5 µL each of the dATP, dCTP, and dGTP stocks.
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3. Mix the nucleotide solution from Step 2 with the 2 M HEPES and the hexadeoxyribonucleotide solution in a ratio of 100:250:150.
- © 2011 Cold Spring Harbor Laboratory Press
