Team:Cambridge/Protocols/DigestionLigation

From 2012.igem.org

(Difference between revisions)
(Digest)
(Ligation)
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===Ligation===
===Ligation===
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:Add 2ul of digested plasmid backbone (25 ng)
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#Add 2ul of digested plasmid backbone (25 ng)
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:Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
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#Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
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:Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
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#Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
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:Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
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#Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
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:Add 0.5 ul T4 DNA ligase
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#Add 0.5 ul T4 DNA ligase
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:Add water to 10 ul
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#Add water to 10 ul
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:Ligate 16C/30 min, heat kill 80C/20 min
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#Ligate 16C/30 min, heat kill 80C/20 min
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:Transform with 1-2 ul of product
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#Transform with 1-2 ul of product
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:Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.
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*Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.

Revision as of 08:46, 19 September 2012

This protocol is adopted from the Parts Registry: http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones. It is used to create biobricks which comply to BBF RFC 10.

Digest

  1. Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns)
    • 5 ul NEB Buffer 2
    • 0.5 ul BSA
    • 0.5 ul EcoRI-HF
    • 0.5 ul PstI
    • 0.5 ul DpnI (Used to digest any template DNA from production)
    • 18 ul dH20
  2. Digest Plasmid Backbone
  3. Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
  4. Add 4 ul of Enzyme Master Mix
  5. Digest 37C/30 min, heat kill 80C/20 min

Ligation

  1. Add 2ul of digested plasmid backbone (25 ng)
  2. Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
  3. Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
  4. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
  5. Add 0.5 ul T4 DNA ligase
  6. Add water to 10 ul
  7. Ligate 16C/30 min, heat kill 80C/20 min
  8. Transform with 1-2 ul of product
  • Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.


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