Team:Wash U/Protocol
From 2009.igem.org
Line 102: | Line 102: | ||
:BioBrick Assembly is a standard protocol for combining two BioBrick parts and positioning them in a destination plasmid. Specific cut sites for standard restriction enzymes are utilized to simplify the protocol and include EcoR1-HF, Xba1, Spe1 and Pst1. The assembly consists of two major parts: digestion and ligation. Note that BioBrick assembly requires purified, isolated DNA for all parts involved (upstream, downstream and destination plasmid). To view the full Biobrick Assembly manual, please click [http://ginkgobioworks.com/support/ here]. | :BioBrick Assembly is a standard protocol for combining two BioBrick parts and positioning them in a destination plasmid. Specific cut sites for standard restriction enzymes are utilized to simplify the protocol and include EcoR1-HF, Xba1, Spe1 and Pst1. The assembly consists of two major parts: digestion and ligation. Note that BioBrick assembly requires purified, isolated DNA for all parts involved (upstream, downstream and destination plasmid). To view the full Biobrick Assembly manual, please click [http://ginkgobioworks.com/support/ here]. | ||
'''Materials''' | '''Materials''' | ||
- | + | * NEBuffer 2 | |
+ | * BSA | ||
+ | * Restriction Enzymes (EcoR1-HF, Xba1, Spe1, Pst1) | ||
+ | * dI H20 | ||
+ | * 10X T4 DNA Ligase Reaction Buffer | ||
+ | * 10X T4 DNA Ligase | ||
+ | * purified upstream BioBrick part | ||
+ | * purified downstream BioBrick part | ||
+ | * purified destination plasmid | ||
'''Procedures''' | '''Procedures''' | ||
# Begin BioBrick Assembly with three separate plasmids, an upstream part, a down stream part, and a destination plasmid. It is important that the destination plasmid contain the toxic gene ccdB in the BioBrick cloning site and a different antibiotic resistance to the upstream and downstream parts. | # Begin BioBrick Assembly with three separate plasmids, an upstream part, a down stream part, and a destination plasmid. It is important that the destination plasmid contain the toxic gene ccdB in the BioBrick cloning site and a different antibiotic resistance to the upstream and downstream parts. |
Revision as of 15:26, 10 July 2009