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Team:UNIPV-Pavia/Protocols/Pcr
From 2008.igem.org
(Difference between revisions)
| Line 45: | Line 45: | ||
**0.6 µl MgCl2 | **0.6 µl MgCl2 | ||
**0.4 µl dNTPs | **0.4 µl dNTPs | ||
| - | **1 µl DNA (or ddH2O for blank sample) | + | **1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution. |
**0.2 µl Taq Polymerase | **0.2 µl Taq Polymerase | ||
**250 nM VF2 primer | **250 nM VF2 primer | ||
**250 nM VR primer | **250 nM VR primer | ||
**A proper amount of ddH2O to have 20 µl of total reaction volume | **A proper amount of ddH2O to have 20 µl of total reaction volume | ||
| - | *into a | + | *into an eppendorf tube. |
| + | *Put the eppendorf tube in the thermal cycler and set this program: | ||
| + | **95°C 10 min (Taq Polymerase activation) | ||
| + | **CYCLE: | ||
| + | ***95°C 30 sec | ||
| + | ***60°C 1 min | ||
| + | ***72°C 1 min | ||
| + | **(end of CYCLE) | ||
| + | **72°C 7 min | ||
| + | **16°C forever. | ||
| + | *Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length. | ||
<br> | <br> | ||
Revision as of 12:41, 21 June 2008
 Home
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|  The Project
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|
|---|---|---|---|---|
 Dry Lab
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|  Protocols
|  Activity Notebook
|
The protocols we used
- LB medium preparation
- Plasmid resuspension from IGEM paper spots
- Transformation
- Plasmid extraction
- BioBrick digestion with restriction enzymes
- DNA gel extraction
- Antarctic Phosphatase
- Ligation
- PCR
Antarctic Phosphatase
(estimated time: 1 hour and 30 min)
Materials needed:
- MgCl2
- Buffer
- dNTPs
- ddH2O
- Taq Polymerase
- VF2 primer
- VR primer
- For every DNA sample you want to amplify, put:
- 2 µl buffer
- 0.6 µl MgCl2
- 0.4 µl dNTPs
- 1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution.
- 0.2 µl Taq Polymerase
- 250 nM VF2 primer
- 250 nM VR primer
- A proper amount of ddH2O to have 20 µl of total reaction volume
- into an eppendorf tube.
- Put the eppendorf tube in the thermal cycler and set this program:
- 95°C 10 min (Taq Polymerase activation)
- CYCLE:
- 95°C 30 sec
- 60°C 1 min
- 72°C 1 min
- (end of CYCLE)
- 72°C 7 min
- 16°C forever.
- Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
