Luckau Protocols:PCR Buffers A-H: Difference between revisions
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==Materials== | ==Materials== | ||
Have the following stock solutions already prepared: | |||
* 1M Tris-Cl, pH 8.3 | * 1M [http://openwetware.org/wiki/Luckau_Protocols:Tris-Cl Tris-Cl], pH 8.3 | ||
* 2.5M KCl | * 2.5M [http://openwetware.org/wiki/Luckau_Protocols:KCl KCl] | ||
* 500mM MgCl<sub>2</sub> | * 500mM [http://openwetware.org/wiki/Luckau_Protocols:MgCl2 MgCl<sub>2</sub>] | ||
Latest revision as of 16:24, 27 September 2012
PCR Buffers A-H |
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
- [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}}{2.5 \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
- In a 100mL glass media bottle, mix the given amount of Tris, KCl and MgCl2 (see above Calculations)
- Add MilliQ water to bring volume to approximately 80 mL
- 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)
- Add MilliQ water to bring to final volume of 100mL
- 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.
- Optional: autoclave solution using the liquid settings.
- Aliquot Buffer A-H into 1mL tubes to be used for PCR at the bench
- 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
-