Luckau Protocols:PCR Buffers A-H

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PCR Buffers A-H

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


Contents

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

Have the following stock solutions already prepared:


Protocol

Calculations

  • 1M Tris-Cl, pH 8.3
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}
  • 2.5M KCl
100 \mathrm{mL} \bullet \tfrac{500 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{2.5 \mathrm{mol}}\ \bullet \tfrac{\mathrm{mol}}{1000 \mathrm{mmol}}\ = 20 \mathrm{mL}
  • 500mM MgCl2
Buffer A: 100 \mathrm{mL} \bullet \tfrac{10 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 2 \mathrm{mL}
Buffer B: 100 \mathrm{mL} \bullet \tfrac{15 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 3 \mathrm{mL}
Buffer C: 100 \mathrm{mL} \bullet \tfrac{20 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 4 \mathrm{mL}
Buffer D: 100 \mathrm{mL} \bullet \tfrac{25 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 5 \mathrm{mL}
Buffer E: 100 \mathrm{mL} \bullet \tfrac{30 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 6 \mathrm{mL}
Buffer F: 100 \mathrm{mL} \bullet \tfrac{35 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 7 \mathrm{mL}
Buffer G: 100 \mathrm{mL} \bullet \tfrac{40 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 8 \mathrm{mL}
Buffer H: 100 \mathrm{mL} \bullet \tfrac{45 \mathrm{mmol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{500 \mathrm{mmol}}\ = 9 \mathrm{mL}


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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