29/07/13

From 2013.igem.org

Revision as of 14:57, 29 July 2013 by Queziatoe (Talk | contribs)
  1. Ran the gel from the 26/07/2013 again for 45min
  2. Digestion of plasmid backbone (pSB1C3)
  • Three different reactions
  • Master Mix (changes were made from the protocol)
    • 5ul NEB Buffer 3.1
    • 0.5ul of EcoRI
    • 0.5ul of PstI
    • 19ul of dH2O
  • For each reaction add:
    • 4ul of linearized backbone
    • 4ul of enzyme master mix
  • digest at 37C for 30min
  • heat kill at 80C for 20min
  1. Digestion of the limonene biobrick plasmid backbone (BBa_K118025)
  • Using clone 1.1 purified plasmid DNA
  • For the reaction adding:
    • 5ul NEB Buffer 3.1
    • 2ul of EcoRI
    • 2ul of PstI
    • 2.5ul of dH2O
    • 38.5ul of DNA
  • digest at 37C for 30min
  • heat kill at 80C for 20min
  1. Ligation of insert to vector
  • Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
  • Add the following substances
    • 11.1ul of dH2O
    • 1ul of QS ligase
    • 5ul of 4xQS buffer (vortex before use)
    • mix thoroughly by pipetting
  • incubate at room temperature for 5 min to create cohesive ends
  • run 2.5-5ul of the ligation mixture onto an agarose gel to check ligation efficiency
  • transform it into competent cells (E.coli)