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Team:Paris Liliane Bettencourt/Notebook/2011/07/22/
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== Cyrille == | == Cyrille == | ||
Prearation of the pHM3 plasmid. This plasmid from H. Pfutzer contains 2 EcoRI excision site. One will be removed by quick change mutagenesis. | Prearation of the pHM3 plasmid. This plasmid from H. Pfutzer contains 2 EcoRI excision site. One will be removed by quick change mutagenesis. | ||
| - | 42 base oligo couple has been ordered from eurogentec. The protocol is the one | + | 42 base oligo couple has been ordered from eurogentec. The protocol is the one [http://labs.fhcrc.org/hahn/Methods/mol_bio_meth/quick_change.htmlhere] using the phusion enzyme. |
| + | |||
| + | The two primers pHM3-fwd, pHM3-rev where resuspended at 1 microM. | ||
| + | The template was mesured with the TECAN and is at 448 ng/microL | ||
| + | |||
| + | The reaction mix contains: | ||
| + | * Buffer HF 5x : 10 microL | ||
| + | * dNTP (2mM) : 5 microL | ||
| + | * Phusion : 1 microL | ||
| + | * Primer fwd : 2 microL | ||
| + | * Primer rev : 3 microL | ||
| + | * Template : 0,7 microL | ||
| + | |||
| + | QSP H2O 50 MicroL | ||
| + | |||
| + | Cycles: | ||
| + | * 98 °C 2 min | ||
| + | |||
| + | * 98°C 10s | ||
| + | * 63°C 1 min | ||
| + | * 72°C 12 min | ||
| + | * Repeat 20 times | ||
| + | |||
| + | * 72°C 10 min | ||
| + | * 4°C forever | ||
| + | |||
| + | The PCR was done overnight and then freeze until monday | ||
| + | |||
| + | |||
| + | ==Mathias & Laura== | ||
| + | |||
| + | ===Miniprep=== | ||
| + | |||
| + | -RFP Standard Terminator RBS & T7 | ||
| + | |||
| + | -GFP mut3b T7 Terminator RBS & T7 | ||
| + | |||
| + | -pT7 I712074 | ||
| + | |||
| + | ===Digestion and gel extraction=== | ||
| + | |||
| + | -RFP Standard Terminator RBS & T7 by Xba1 and Pst1 | ||
| + | |||
| + | -GFP mut3b T7 Terminator RBS & T7 by Xba1 and Pst1 | ||
| + | |||
| + | -pT7 I712074 by Spe1 and Pst1 | ||
| + | |||
| + | -pVeg + SpoVg and LacI + Term by Spe1 and Pst1 | ||
| + | |||
| + | ===Ligation and transformation=== | ||
| + | |||
| + | -GFP mut3b T7 Terminator RBS T7 & pT7 => T7 Autoamplifier system + Reporter K606036 | ||
Latest revision as of 16:20, 21 September 2011
Contents |
Cyrille
Prearation of the pHM3 plasmid. This plasmid from H. Pfutzer contains 2 EcoRI excision site. One will be removed by quick change mutagenesis.
42 base oligo couple has been ordered from eurogentec. The protocol is the one [http://labs.fhcrc.org/hahn/Methods/mol_bio_meth/quick_change.htmlhere] using the phusion enzyme.
The two primers pHM3-fwd, pHM3-rev where resuspended at 1 microM. The template was mesured with the TECAN and is at 448 ng/microL
The reaction mix contains:
- Buffer HF 5x : 10 microL
- dNTP (2mM) : 5 microL
- Phusion : 1 microL
- Primer fwd : 2 microL
- Primer rev : 3 microL
- Template : 0,7 microL
QSP H2O 50 MicroL
Cycles:
- 98 °C 2 min
- 98°C 10s
- 63°C 1 min
- 72°C 12 min
- Repeat 20 times
- 72°C 10 min
- 4°C forever
The PCR was done overnight and then freeze until monday
Mathias & Laura
Miniprep
-RFP Standard Terminator RBS & T7
-GFP mut3b T7 Terminator RBS & T7
-pT7 I712074
Digestion and gel extraction
-RFP Standard Terminator RBS & T7 by Xba1 and Pst1
-GFP mut3b T7 Terminator RBS & T7 by Xba1 and Pst1
-pT7 I712074 by Spe1 and Pst1
-pVeg + SpoVg and LacI + Term by Spe1 and Pst1
Ligation and transformation
-GFP mut3b T7 Terminator RBS T7 & pT7 => T7 Autoamplifier system + Reporter K606036
