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Revision as of 07:55, 18 August 2013 (view source) EstherJ (Talk | contribs) ← Older edit		Revision as of 22:57, 26 August 2013 (view source) E.Soboslay (Talk | contribs) (→Procedure) Newer edit →
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	== Procedure ==		== Procedure ==
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		+
		+	1. Add 2µL of digested plasmid backbone
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		+	2. Add equimolar amount of digested Part 1
		+
		+	3. Add equimolar amount of digested Part 2
		+
		+	4. Add 1µL T4 DNA ligase buffer.
		+
		+	5. Add 0.5µL T4 DNA ligase.
		+
		+	6. Add water to 10µL.
		+
		+	7. Place in pre-programmed thermal cycler at 16°C for 30 minutes then at 80°C for 20 minutes.

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Revision as of 22:57, 26 August 2013

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Ligation Protocol

Protocol from: http://parts.igem.org/Help:Protocols/Ligation

Materials

  -digested plasmid backbone
  -equimolar amount of EcoRI-HF SpeI digested fragment
  -equimolar amount of XbaI PstI digested fragment
  -T4 DNA ligase buffer
  -T4 DNA ligase
  -dH2O

Procedure

1. Add 2µL of digested plasmid backbone

2. Add equimolar amount of digested Part 1

3. Add equimolar amount of digested Part 2

4. Add 1µL T4 DNA ligase buffer.

5. Add 0.5µL T4 DNA ligase.

6. Add water to 10µL.

7. Place in pre-programmed thermal cycler at 16°C for 30 minutes then at 80°C for 20 minutes.