User:Karmella Haynes/Notebook/BioBrick cloning/2015/01/22

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01/22/15

  • Gal4 fusion cloning


Planning: Gal4 fusion cloning

  • "Backbone" = Gal4-mCherry-VP64 construct KAH60/pcVN
  • Approach: swap-in activator domain to replace VP64
    • PCR-amplify backbone (omitting VP64)
    • PCR-amplify insert
    • Plan A: use LCR to ligate insert into backbone


Primers (ordered 01/02/15)
Attempt 1: swap-in ATF activation domain; add 5' phosphates to all oligos EXCEPT for bridging oligos

  • KAH60 "backbone" (without VP64, includes scars) = 6637
    • KAH60 F1
    • KAH60 R1
  • ATF2 insert = 906
    • ATF2_f1
    • ATF2_r1
  • LCR Bridging oligos
    • LCRb_KAH60_ATF2_1
    • LCRb_KAH60_ATF2_2


PCR-verify fragment primers
Try different cDNA libraries for the ATF2 fragment to account for any mutations or deletions

  1. KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1
  2. U2OS C002; ATF2 insert = 906: ATF2_f1/ ATF2_r1
  3. SKNSH C001; same
  4. K562 C001; same


Reagent Rxn1 Rxn2 Rxn3 Rxn4 Hover name
30 μL/lane, 1% agarose; Ladder
Template 0.2 KAH60/pcVN 1.0 1:1000 U2OS cDNA 1.0 1:1000 SKNSH cDNA 1.0 1:1000 K562 cDNA
10 uM fwd primer 1.0 1.0 1.0 1.0
10 uM rev primer 1.0 1.0 1.0 1.0
2x GoTaq green 12.5 12.5 12.5 12.5
dH2O 10.3 9.5 9.5 9.5
  25.0 25.0 25.0 25.0

Program: GOTAQ35cyc

  • 95°C, 3 min
  • 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
  • 72°C, 3 min
  • 4°C ∞



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