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Team:EPF-Lausanne/Protocols/PCR
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==Primers== | ==Primers== | ||
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===Binding DNA=== | ===Binding DNA=== | ||
'''1.''' Add 4 volumes of PureLink™ Binding Buffer with isopropanol (above) or Binding Buffer HC with isopropanol (above) to 1 volume of PCR (50-100 μl). Mix well. | '''1.''' Add 4 volumes of PureLink™ Binding Buffer with isopropanol (above) or Binding Buffer HC with isopropanol (above) to 1 volume of PCR (50-100 μl). Mix well. | ||
| - | |||
'''2.''' Remove a PureLink™ Spin Column in a Collection Tube from the package. | '''2.''' Remove a PureLink™ Spin Column in a Collection Tube from the package. | ||
| - | |||
'''3.''' Add sample with appropriate Binding Buffer from Step 1 to the PureLink™ Spin Column. | '''3.''' Add sample with appropriate Binding Buffer from Step 1 to the PureLink™ Spin Column. | ||
| - | |||
'''4.''' Centrifuge the column at room temperature at 10,000 × g for 1 minute. | '''4.''' Centrifuge the column at room temperature at 10,000 × g for 1 minute. | ||
| - | |||
'''5.''' Discard the flow through and place the spin column into the collection tube. | '''5.''' Discard the flow through and place the spin column into the collection tube. | ||
| - | |||
'''6.''' Proceed to Washing DNA, next protocol. | '''6.''' Proceed to Washing DNA, next protocol. | ||
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===Washing DNA=== | ===Washing DNA=== | ||
| - | '''1.''' Add 650 μl of Wash Buffer with ethanol | + | '''1.''' Add 650 μl of Wash Buffer with ethanol to the column. |
| - | + | ||
'''2.''' Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube. | '''2.''' Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube. | ||
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'''3.''' Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube. | '''3.''' Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube. | ||
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'''4.''' Proceed to Eluting DNA, below. | '''4.''' Proceed to Eluting DNA, below. | ||
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===Eluting DNA=== | ===Eluting DNA=== | ||
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'''1.''' Place the spin column in a clean 1.7-ml PureLink™ Elution Tube supplied with the kit. | '''1.''' Place the spin column in a clean 1.7-ml PureLink™ Elution Tube supplied with the kit. | ||
| - | + | '''2.''' Add 30 μl of Elution Buffer (10 mM Tris-HCl, pH 8.5) or sterile, distilled water (pH >7.0) to the center of the column. Here we purify the 2 tubes in one column. | |
| - | '''2.''' Add | + | |
| - | + | ||
'''3.''' Incubate the column at room temperature for 1 minute. | '''3.''' Incubate the column at room temperature for 1 minute. | ||
| - | |||
'''4.''' Centrifuge the column at maximum speed for 2 minutes. | '''4.''' Centrifuge the column at maximum speed for 2 minutes. | ||
| - | |||
'''5.''' The elution tube contains your purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μl. | '''5.''' The elution tube contains your purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μl. | ||
| - | |||
'''6.''' Store the purified PCR product at -20°C or use PCR product for the desired downstream application. | '''6.''' Store the purified PCR product at -20°C or use PCR product for the desired downstream application. | ||
Latest revision as of 18:40, 20 October 2009
Primers
Make primers pair mix with 3µM concentration (total).
| Plasmid | 0.5ul |
|---|---|
| Primers pair (3µM) | 1ul |
| Thermo POL buffer | 2.5µl |
| dNTP | 0.5 µl |
| Taq EPFL polym | 0.25 µl |
| H20 (MQ) | 20.25 µl |
| Total | 24.5 µl |
We used 2 tubes of PCR of 25µl each.
Cycles
Program PCR: count 1min/kb
- 94°C – 120’’
- 94°C – 45’’ |
- 56°C – 45’’ | → 30x
- 68°C – 120’’ |
- 72°C – 420’' |
- 10°C – ∞
PCR purification
PureLink™ PCR Purification Kit – Invitrogen
Binding DNA
1. Add 4 volumes of PureLink™ Binding Buffer with isopropanol (above) or Binding Buffer HC with isopropanol (above) to 1 volume of PCR (50-100 μl). Mix well.
2. Remove a PureLink™ Spin Column in a Collection Tube from the package.
3. Add sample with appropriate Binding Buffer from Step 1 to the PureLink™ Spin Column.
4. Centrifuge the column at room temperature at 10,000 × g for 1 minute.
5. Discard the flow through and place the spin column into the collection tube.
6. Proceed to Washing DNA, next protocol.
Washing DNA
1. Add 650 μl of Wash Buffer with ethanol to the column.
2. Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube.
3. Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube.
4. Proceed to Eluting DNA, below.
Eluting DNA
1. Place the spin column in a clean 1.7-ml PureLink™ Elution Tube supplied with the kit.
2. Add 30 μl of Elution Buffer (10 mM Tris-HCl, pH 8.5) or sterile, distilled water (pH >7.0) to the center of the column. Here we purify the 2 tubes in one column.
3. Incubate the column at room temperature for 1 minute.
4. Centrifuge the column at maximum speed for 2 minutes.
5. The elution tube contains your purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μl.
6. Store the purified PCR product at -20°C or use PCR product for the desired downstream application.
