Team:BCCS-Bristol/Wetlab/K381001 Construction

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==Constructing Our Biobrick - K381001==
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Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick.
Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick.

Revision as of 17:01, 22 September 2010

Constructing Our Biobrick - K381001

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