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| + | [[Paris/July 31|yesterday]] -- [[Paris/August 2|tomorrow]] <br> | ||
== DGAT Assembly PCR: Megaprimer PCR == | == DGAT Assembly PCR: Megaprimer PCR == | ||
| - | Material: | + | * Material: <br> |
| - | - DNA plasmid (pKS::DGAT): 1.5µL (200ng) | + | - DNA plasmid (pKS::DGAT): 1.5µL (200ng)<br> |
| - | - Tampon: 10µL | + | - Tampon: 10µL<br> |
| - | - dNTP: 1µL (final 200µmol each) | + | - dNTP: 1µL (final 200µmol each)<br> |
| - | - Taq: 0.5µL | + | - Taq: 0.5µL<br> |
| - | - Water: 29µL | + | - Water: 29µL<br> |
| - | - Primer: ForDGAT1: 4µL (final 0.8µM) | + | - Primer: ForDGAT1: 4µL (final 0.8µM)<br> |
| - | - Megaprimer: DGAT2: 4µL ( | + | - Megaprimer: DGAT2: 4µL (50ng)<br> |
| + | |||
| + | [[Image: DGATDPst108012007.jpg|right|100px]] | ||
| + | |||
| + | * PCR Program:<br> | ||
| + | |||
| + | 5 cycles only with DGAT2 as primer:<br> | ||
| + | - 98°C: 5min<br> | ||
| + | - 98°C: 1min (X5)<br> | ||
| + | - 60°C: 30s (X5)<br> | ||
| + | - 72°C: 2min (X5)<br> | ||
| + | - 72°C: 10 min<br> | ||
| + | - 4°C: inf | ||
| + | <br> | ||
| + | |||
| + | Add primer ForDGAT1: | ||
| + | |||
| + | 30 cycles: | ||
| + | - 98°C: 5min<br> | ||
| + | - 98°C: 1min (X5)<br> | ||
| + | - 60°C: 30s (X5)<br> | ||
| + | - 72°C: 2min (X5)<br> | ||
| + | - 72°C: 10 min<br> | ||
| + | - 4°C: inf | ||
| + | |||
| + | * '''Quantity:''' 5ng/µL <br> | ||
| + | * '''Expected size:''' 1443 bp | ||
| + | |||
| + | * After purification: <br> | ||
| + | [[Image: DGATDPst108012007purification.jpg|right|100px]] | ||
| + | 2µL of purification product (2µL of ladder): <br> | ||
| + | - well 0: ladder<br> | ||
| + | - well 1: negative control of purification<br> | ||
| + | - well 2: purification product<br> | ||
Latest revision as of 17:52, 7 October 2007
DGAT Assembly PCR: Megaprimer PCR
- Material:
- DNA plasmid (pKS::DGAT): 1.5µL (200ng)
- Tampon: 10µL
- dNTP: 1µL (final 200µmol each)
- Taq: 0.5µL
- Water: 29µL
- Primer: ForDGAT1: 4µL (final 0.8µM)
- Megaprimer: DGAT2: 4µL (50ng)
- PCR Program:
5 cycles only with DGAT2 as primer:
- 98°C: 5min
- 98°C: 1min (X5)
- 60°C: 30s (X5)
- 72°C: 2min (X5)
- 72°C: 10 min
- 4°C: inf
Add primer ForDGAT1:
30 cycles:
- 98°C: 5min
- 98°C: 1min (X5)
- 60°C: 30s (X5)
- 72°C: 2min (X5)
- 72°C: 10 min
- 4°C: inf
- Quantity: 5ng/µL
- Expected size: 1443 bp
- After purification:
2µL of purification product (2µL of ladder):
- well 0: ladder
- well 1: negative control of purification
- well 2: purification product
