Week 6 Lab: Assembly

PCR Calcs

PCR Setup Purple and Orange

  • NFW – added
  • fwd - Pu, added
  • fwd - O, added
  • rev – same for both, added
  • Master mix – added
  • Template plasmid – added

Calculated PCR params and started running

Need to add DPN1 to digest the template DNA from the PCRs for w/out template

  • DPN1 currently at -20 BEL – thaw for a bit

  • 1 ul DPN1 to ea rxn (put back into freezer)

  • incubate 37 45 m or so – standing inc

  • DPN1

  • PCR Cleanup

  • Run gel

PCR Cleanup

  1. Dilute samples with DNA Binding Buffer. Add buffer in a 2:1 ratio of binding buffer to sample for fragments larger than 2kb (e.g. add 54µl Binding buffer to 27µl DNA). For fragments smaller than 2kb add buffer in a 5:1 ratio (e.g. add 135µl Binding buffer to 27µl DNA).
  • add 50 to backbone
  • add 125 ul to others