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From 2013.igem.org

Revision as of 14:10, 29 July 2013 (view source) Queziatoe (Talk | contribs) ← Older edit		Revision as of 14:54, 29 July 2013 (view source) Queziatoe (Talk | contribs) Newer edit →
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	*digest at 37C for 30min		*digest at 37C for 30min
	*heat kill at 80C for 20min		*heat kill at 80C for 20min
		+
		+	#Ligation of insert to vector
		+	*Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
		+	*Add the following substances
		+	**11.1ul of dH2O
		+	**1ul of QS ligase
		+	**5ul of 4xQS buffer (vortex before use)
		+	**mix

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Revision as of 14:54, 29 July 2013

1. Ran the gel from the 26/07/2013 again for 45min
2. Digestion of plasmid backbone (pSB1C3)

• Three different reactions
• Master Mix (changes were made from the protocol)
  • 5ul NEB Buffer 3.1
  • 0.5ul of EcoRI
  • 0.5ul of PstI
  • 19ul of dH2O
• For each reaction add:
  • 4ul of linearized backbone
  • 4ul of enzyme master mix
• digest at 37C for 30min
• heat kill at 80C for 20min

1. Digestion of the limonene biobrick plasmid backbone (BBa_K118025)

• Using clone 1.1 purified plasmid DNA
• For the reaction adding:
  • 5ul NEB Buffer 3.1
  • 2ul of EcoRI
  • 2ul of PstI
  • 2.5ul of dH2O
  • 38.5ul of DNA
• digest at 37C for 30min
• heat kill at 80C for 20min

1. Ligation of insert to vector

• Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
• Add the following substances
  • 11.1ul of dH2O
  • 1ul of QS ligase
  • 5ul of 4xQS buffer (vortex before use)
  • mix