IGEM:Peking University/2008/Experiment/PKU 0620 G2 1: Difference between revisions
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(New page: ==Primer design== :'''Primer 1''' :*Primer name: F-HindIII-Apal-Gal4 :*Sequence(5'to3'):CCCAAGCTTGGGCCCAAGATGAAGCTACTGTCTTCTATCGAACAA :*Tm:64.9°C :'''Primer 2''' :*Primer name: R-EcoRI-Sp...) |
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Revision as of 05:19, 4 July 2008
Primer design
- Primer 1
- Primer name: F-HindIII-Apal-Gal4
- Sequence(5'to3'):CCCAAGCTTGGGCCCAAGATGAAGCTACTGTCTTCTATCGAACAA
- Tm:64.9°C
- Primer 2
- Primer name: R-EcoRI-Spel-Gal4
- Sequence(5'to3'):GGGGAATTCACTAGTATTTTACTCTTTTTTTGGGTTTGGTGGGGT
- Tm:61.3°C
PCR
| Volume (μL) | |
| Template （pPT1） | 0.5 |
| dNTP | 1 |
| Enzyme | 0.5 |
| Buffer | 10 |
| F-HindIII-Apal-Gal4 | 0.5 |
| R-EcoRI-Spel-Gal4 | 0.5 |
| ddH2O | 37 |
- conditions
| Temperature | Time Length(0:00:00) | |
| 98°C | 00:30 | |
| 98°C | 00:10 | 30 cycles |
| 57.1,60.4,62.5,64.7,66.9,68.9°C | 00:30 | 30 cycles |
| 72°C | 01:30 | 30 cycles |
| 72°C | 07:00 | |
| 4°C | Hold |
- Enzyme cutting-DNA (pGREG503/504)-SacI-XhoI-20 μL
| Volumn(μL) | ||
| pGREG503/504 | 5 | |
| SacI | 0.2 | |
| XhoI | 0.2 | *5 |
| M Buffer | 2 | |
| ddH2O | 12.6 |
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