User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/16

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Possible BioBrick Cloning Strategy

Should Dr. Haynes' insertion of blunt site into MV10 for LCR not be effective, the following BioBrick strategy could be used to build desired activator domain constructs:

STAGE 1 – Reconstruct MV10 with Gal4DB_mCh domain

  • Design two single stranded oligos with Kozaks sequence followed by Xba/Spe site for insertion after CMV and before NLS
  • Digest MV10 plasmid Spe/Xba, add oligos and PNK to ligate
  • Amplify Gal4DB_mCh domain with BB suffix and prefix via PCR
  • Insert into MV10 Xba/Spe site

STAGE 2 – Activator Domains

  • Mutate restriction sites out of the following activators:
    • ATF2: one EcoRI site, one PstI site, one XbaI site
    • p300: one XbaI site, three PstI
    • CBP: three PstI sites
    • KMT2A: one PstI site
    • ANM5: one PstI site
    • SMCA4: two EcorI sites, one NotI site, ten PstI sites
  • PCR amplify activators with BB suffix and prefix

STAGE 3 – Build Constructs

  • Insert activator domains after Gal4DB_mCh