Team:BCCS-Bristol/Wetlab/K381001 Construction


Revision as of 11:33, 17 September 2010 by Kcoyte (Talk | contribs)

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
  • 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