"
Team:EPF-Lausanne/Protocols/PCR
From 2009.igem.org
(→Cycles) |
|||
| (10 intermediate revisions not shown) | |||
| Line 15: | Line 15: | ||
<html><center> | <html><center> | ||
| - | <font size="6" color="#007CBC" | + | <font size="6" color="#007CBC">PCR protocol</font> |
</center></html> | </center></html> | ||
<br> | <br> | ||
| - | + | ||
| - | + | ||
| - | + | ||
==Primers== | ==Primers== | ||
Make primers pair mix with 3µM concentration (total). | Make primers pair mix with 3µM concentration (total). | ||
| - | {| class="wikitable" style="text-align:center; width: | + | {| class="wikitable" style="text-align:center; width:50%;" |
|+ '''<big>Reaction mix</big>''' | |+ '''<big>Reaction mix</big>''' | ||
|- | |- | ||
| Line 49: | Line 47: | ||
|'''24.5 µl''' | |'''24.5 µl''' | ||
|} | |} | ||
| + | |||
| + | |||
| + | We used '''2''' tubes of PCR of 25µl each. | ||
==Cycles== | ==Cycles== | ||
| Line 54: | Line 55: | ||
:94°C – 120’’ | :94°C – 120’’ | ||
| - | :94°C – 45’’ | | + | :94°C – 45’’ | |
| - | :56°C – 45’’ | → 30x | + | :56°C – 45’’ | → 30x |
:68°C – 120’’ | | :68°C – 120’’ | | ||
:72°C – 420’' | | :72°C – 420’' | | ||
| Line 61: | Line 62: | ||
| + | ==PCR purification== | ||
| + | |||
| + | <big> PureLink™ PCR Purification Kit – Invitrogen</big> | ||
| + | |||
| + | |||
| + | ===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. | ||
</div><div CLASS="epfl09bouchon"></div> | </div><div CLASS="epfl09bouchon"></div> | ||
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.
