OVERVIEW
Here we describe a procedure to prepare 10 ml of Potassium chloride-based 10x PCR buffer. The 10x buffer contains 500 mM KCl, 100 mM Tris.Cl (pH 9.0), 15 mM MgCl2, and 1% Triton X-100. The buffer is prepared by mixing 5 ml of 1 M KCl, 1 ml of 1 M Tris.Cl (pH 9.0 at 25°C), 150 µl of 1 M MgCl2, and 0.5 ml of 20% Triton X-100 in water to a final volume of 10 ml.
REQUIREMENTS
Reagents and solutions
♦ 1 M KCl
♦ 1 M Tris.Cl (pH 9.0 at 25°C)
♦ 1 M MgCl2
♦ 20% Triton X 100
♦ MiliQ water (autoclaved sterile)
Equipment and disposables
♦ Micropipette and tips
♦ Falcon® screw cap graduated Conical Tubes, 15 mL (e.g., SARSTEDT, 62.554.502)
COMPOSITION (10x Buffer)
♦ 500 mM KCl
♦ 100 mM Tris.Cl (pH 9.0 at 25°C)
♦ 15 mM MgCl2
♦ 1% Triton X-100
OBJECTIVE
Preparation of 10 ml of Potassium chloride-based 10x PCR buffer
PROCEDURE
Use appropriate personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow your institute’s guidelines.
Step 1: Step 1: Take 3.35 ml water in a 15 ml tube. Now add 5 ml of 1 M KCl, 1 ml of 1 M Tris.Cl, 0.5 ml of 20% Triton X 100, and 150 µl of 1 M MgCl2.
Precaution:
Wear gloves and use high-quality molecular biology-grade reagents and water (e.g., MilliQ water). The prepared buffer must be free of any nucleic acid and nucleases.
Step 2: Mix the solution by inverting the tube a few times.
STORAGE
Store the buffer in the freezer (-20°C) in small aliquots.
REFERENCES:
