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Revision as of 14:37, 2 August 2007 (view source) Davidpz (Talk | contribs) (→DGAT Assembly PCR: Megaprimer PCR) ← Older edit		Revision as of 10:06, 3 August 2007 (view source) Davidpz (Talk | contribs) (→DGAT Assembly PCR: Megaprimer PCR) Newer edit →
Line 34:		Line 34:
	- 4°C: inf		- 4°C: inf
-	* '''Quantity:''' 5µg/µL <br>	+	* '''Quantity:''' 5ng/µL <br>
	* '''Expected size:''' 1443 bp		* '''Expected size:''' 1443 bp

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Revision as of 10:06, 3 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)

• 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