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Team:UNIPV-Pavia/Protocols/Pcr
From 2008.igem.org
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*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | ||
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]] | *[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]] | ||
*[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]] | *[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]] | ||
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]] | *[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]] | ||
| Line 27: | Line 28: | ||
| - | <h1> | + | <h1>PCR</h1> |
| - | ''(estimated time: | + | ''(estimated time: 3 hours and 30 min)'' |
<br> | <br> | ||
<br> | <br> | ||
| Line 52: | Line 53: | ||
*into an eppendorf tube. | *into an eppendorf tube. | ||
*Put the eppendorf tube in the thermal cycler and set this program: | *Put the eppendorf tube in the thermal cycler and set this program: | ||
| - | **95°C 10 min | + | **95°C 10 min |
**CYCLE: | **CYCLE: | ||
***95°C 30 sec | ***95°C 30 sec | ||
***60°C 1 min | ***60°C 1 min | ||
| - | ***72°C 1 min | + | ***72°C 1-3 min |
| - | ** | + | **for 35 cycles |
**72°C 7 min | **72°C 7 min | ||
**16°C forever. | **16°C forever. | ||
*Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length. | *Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length. | ||
<br> | <br> | ||
Latest revision as of 10:16, 27 July 2008
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The protocols we used
- LB medium preparation
- Plasmid resuspension from IGEM paper spots
- Transformation
- Plasmid extraction
- BioBrick digestion with restriction enzymes
- DNA gel extraction
- DNA precipitation with sodium acetate
- Antarctic Phosphatase
- Ligation
- PCR
PCR
(estimated time: 3 hours 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
- CYCLE:
- 95°C 30 sec
- 60°C 1 min
- 72°C 1-3 min
- for 35 cycles
- 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.
