Luckau Protocols:PCR Buffers A-H

PCR Buffers A-H

Tara K. Luckau's Home Page Conservation Genetics Lab Notebook Tara's Protocols

Purpose

• PCR Buffer provides a stable chemical environment for polymerase during PCR. The buffer contains Tris (pH buffer, because DNA hydrolyzes in acidic conditions), MgCl₂ (the divalent cation Mg++ is a cofactor for polymerase; the positively charged Mg++ stabilizes negatively charged DNA) and KCl (the positively charged K+ stabilizes negatively charged DNA).
• In the Clark Lab, a series of eight buffers of varying magnesium chloride concentrations is used to optimize PCR conditions.

Info

Buffer Name	Tris	KCl	MgCl2
Buffer A	100 mM, pH 8.3	500 mM	10 mM
Buffer B	100 mM, pH 8.3	500 mM	15 mM
Buffer C	100 mM, pH 8.3	500 mM	20 mM
Buffer D	100 mM, pH 8.3	500 mM	25 mM
Buffer E	100 mM, pH 8.3	500 mM	30 mM
Buffer F	100 mM, pH 8.3	500 mM	35 mM
Buffer G	100 mM, pH 8.3	500 mM	40 mM
Buffer H	100 mM, pH 8.3	500 mM	45 mM

Materials

• 1M Tris-Cl, pH 8.3
• 2.5M KCl
• 500mM MgCl₂

Protocol

Calculations

• 1M Tris-Cl, pH 8.3

[math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{100 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{1 \mathrm{mol}}\ \bullet \tfrac{\mathrm{mol}}{1000 \mathrm{mmol}}\ = 10 \mathrm{mL} }[/math]

• 2.5M KCl

[math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{500 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{5 \mathrm{mol}}\ \bullet \tfrac{\mathrm{mol}}{1000 \mathrm{mmol}}\ = 20 \mathrm{mL} }[/math]

• 500mM MgCl₂

Buffer A: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{10 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 2 \mathrm{mL} }[/math]

Buffer B: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{15 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 3 \mathrm{mL} }[/math]

Buffer C: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{20 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 4 \mathrm{mL} }[/math]

Buffer D: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{25 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 5 \mathrm{mL} }[/math]

Buffer E: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{30 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 6 \mathrm{mL} }[/math]

Buffer F: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{35 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 7 \mathrm{mL} }[/math]

Buffer G: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{40 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 8 \mathrm{mL} }[/math]

Buffer H: [math]\displaystyle{ 100 \mathrm{mL} \bullet \tfrac{45 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 9 \mathrm{mL} }[/math]

To make 100 mL of each 10x PCR Buffer

1. In a 100mL glass media bottle, mix the given amount of Tris, KCl and MgCl₂ (see above Calculations)
2. Add MilliQ water to bring volume to approximately 80 mL
3. Adjust the pH to 8.3 (as with all pH adjustments, be patient - adjust to 8.3, then leave for an hour or so, then re-check the pH)
4. Add MilliQ water to bring to final volume of 100mL
5. You can re-check the pH again, but it should be fine. It's not a bad idea to re-re-check the pH the following day.
6. Optional: autoclave solution using the liquid settings.
7. Aliquot Buffer A-H into 1mL tubes to be used for PCR at the bench
8. Store stock Buffer A-H in the refrigerator to prevent evaporation (and thus pH changes)

Sources

• These buffers were originally formulated as "house buffers" at the University of Arizona Genetics Core, personal communication

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