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Team:BCCS-Bristol/Wetlab/K381001 Construction
From 2010.igem.org
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* Generating larger quantities of both our component biobricks | * Generating larger quantities of both our component biobricks | ||
* Digesting each of these biobricks | * Digesting each of these biobricks | ||
| + | * Phosphatase treatment of the vector and gel extraction of the digests | ||
* Ligating together the desired parts to create a new biobrick | * Ligating together the desired parts to create a new biobrick | ||
* Generating larger quantities of this biobrick, checking and sequencing | * Generating larger quantities of this biobrick, checking and sequencing | ||
Revision as of 11:36, 17 September 2010
Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick.
BBa_I13522 was taken from well 8A kitplate 2 of the 2010 distribution BBa_K216005 was requested from the parts registry
Construction of the biobrick can be broadly broken down into
- Generating larger quantities of both our component biobricks
- Digesting each of these biobricks
- Phosphatase treatment of the vector and gel extraction of the digests
- Ligating together the desired parts to create a new biobrick
- Generating larger quantities of this biobrick, checking and sequencing
Generating large quantities of DNA:
- The first stage was to transform commercial NovaBlue competent cells with our component biobricks, typically transforming 50μL of cells with 2μL of DNA
- Colonies from these transformations were then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
- Combined 3mL of BBa_K216005, BBa_E0840 and BBa_E0240 cell cultures in 1.5mL eppendorf tubes twice - total of 6mL combined cell culture for each biobrick. Performed a miniprep on the combined cultures using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 100μL of solution containing BBa_K216005, BBa_E0840 and BBa_E0240 biobricks in high concentration.
Digesting Component Biobricks:
- BBa_E0840 and BBa_E0240 were digested with Xba1 and Pst1 using the following mix:
- 30μL DNA
- 0.5μL BSA 100x
- 5μL NEB Buffer 3
- 1μL Pst1
- 1.3μL Xba1
- 12.2μL ddH2O
- BBa_K216005 was digested with Spe1 and Pst1 using the following mix:
- 30μL DNA
- 0.5μL BSA 100x
- 5μL NEB Buffer 2
- 1.3μL Pst1
- 1μL Spe1
- 12.2μL ddH2O
- Each digest was left for 2 hours at 37°C
