Paris/August 1

From 2007.igem.org

(Difference between revisions)
(DGAT Assembly PCR: Megaprimer PCR)
(DGAT Assembly PCR: Megaprimer PCR)
Line 38: Line 38:
* After purification: <br>
* After purification: <br>
-
2µL of purification product (2µL of ladder)
+
2µL of purification product (2µL of ladder): <br>
 +
- well 0: ladder
 +
- well 1: negative control of purification
 +
- well 2: purification product
[[Image: DGATDPst108012007purification.jpg|right|100px]]
[[Image: DGATDPst108012007purification.jpg|right|100px]]

Revision as of 14:36, 2 August 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)

DGATDPst108012007.jpg
  • 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: 5µg/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

DGATDPst108012007purification.jpg