"
Team:UNIPV-Pavia/Protocols/Pcr
From 2008.igem.org
(Difference between revisions)
| Line 57: | Line 57: | ||
***95°C 30 sec | ***95°C 30 sec | ||
***60°C 1 min | ***60°C 1 min | ||
| - | ***72°C 1 min | + | ***72°C 1-2 min |
**for 35 cycles (40 if you are performing a colony PCR) | **for 35 cycles (40 if you are performing a colony PCR) | ||
**72°C 7 min | **72°C 7 min | ||
Revision as of 21:25, 5 July 2008
 Home
|  The Team
|  The Project
|  Biological Safety
|  Parts Submitted to the Registry
|
|---|---|---|---|---|
 Dry Lab
|  Wet Lab
|  Modeling
|  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
- 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 1-2 min
- for 35 cycles (40 if you are performing a colony PCR)
- 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.
