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Team:Cambridge/Protocols/DigestionLigation
From 2012.igem.org
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#Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns) | #Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns) | ||
| - | *5 ul NEB Buffer 2 | + | :*5 ul NEB Buffer 2 |
| - | *0.5 ul BSA | + | :*0.5 ul BSA |
| - | *0.5 ul EcoRI-HF | + | :*0.5 ul EcoRI-HF |
| - | *0.5 ul PstI | + | :*0.5 ul PstI |
| - | *0.5 ul DpnI (Used to digest any template DNA from production) | + | :*0.5 ul DpnI (Used to digest any template DNA from production) |
| - | *18 ul dH20 | + | :*18 ul dH20 |
#Digest Plasmid Backbone | #Digest Plasmid Backbone | ||
#Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total) | #Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total) | ||
Revision as of 08:45, 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
- 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
- Digest Plasmid Backbone
- Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
- Add 4 ul of Enzyme Master Mix
- Digest 37C/30 min, heat kill 80C/20 min
Ligation
- Add 2ul of digested plasmid backbone (25 ng)
- Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
- Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
- Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
- Add 0.5 ul T4 DNA ligase
- Add water to 10 ul
- Ligate 16C/30 min, heat kill 80C/20 min
- 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.
