Team:UCLA/Notebook/Honeybee Silk/26 April 2015

iGEM UCLA




4/26 PCR off honey bee gene block
  • See Benchling for the g block sequence and primer reference code https://benchling.com/s/p7bClpzQ/edit
  • The goal of this is to clone 2 different constructs and prepare them as biobricks, one that is just the silk gene, and the other that as the promoter, rbs, etc.. required for protein expression.
  • Using primers p3, p7, and p8. (See Benchling link)

  • PCR Reaction 1 (q5 polymerase kit): primers honeybee p#7 and honeybee p#8 with gBlock, and honeybee p#3 and honeybee p#8 amplify just the honeybee coding region and the prom + coding region respectively

    Component Volume
    5X Q5 Reaction Buffer 5
    10 mM dNTPs 0.5
    10 uM Forward (primer 3/7) 1.25
    10 uM Reverse (primer 8) 1.25
    Template (diluted to 1ng/uL) 0.5
    Q5 High Fidelity DNA Polymerase 0.25
    Nuclease Free Water 16.25
  • PCR program (using benchling annealing temperatures)
    Temperature (C) Time (Min:sec)
    98 0:30
    98 x 30 cycles 0:10
    grad. 55.4/58.4/61.4 C x 30 0:20
    72 x 30 0:30
    72(diluted to 1ng/uL) 2:00
    12 hold

    • The program and pcr reaction recipe were based on the manufacturer protocol. These annealing temperatures were determined using benchling's algorithm. In the future we will use the NEB annealing temperature calculator, because it is more accurate and takes the type of polymerase into account See tomorrow's entry for the gel image of the pcr reaction