Team:Cambridge/Protocols/DigestionLigation

Making a Biobrick

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 and insert (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. To each backbone tube, add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
3. To each insert tube, add an equimolar quantity of insert fragment.
4. To each tube, add 4 ul of Enzyme Master Mix
5. Digest 37C/30 min, heat kill 80C/20 min
6. Your backbone is now ready to receive its insert.

Tips

To prevent self-ligation of backbone, alkaline phosphatase treatment is recommended before ligation. The protocol could be found here: http://openwetware.org/wiki/Phosphatase_treatment_of_linearized_vector (The lab uses USB shrimp alkaline phosphatase)

Ligation

1. Add 2ul of digested plasmid backbone (25 ng)
2. Add equimolar amount of EcoRI and PstI digested insert (< 3 ul).
3. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
4. Add 0.5 ul T4 DNA ligase.
5. Add water to 10 ul.
6. Ligate 16C/30 min, heat kill 80C/20 min.
7. 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.

Tips

• If designing primers to PCR your insert into a biobrick (by adding prefix and suffix tails to your sequence), be sure to add around 10bp of junk at the end of the prefix and suffix to help the restriction endonuclease to cut at the end of the prefix/suffix.
• Make sure that your insert does not contain EcoRI or PstI sites before using this protocol. You may have to perform site directed mutagenesis to make sure that this is not an issue.