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

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Karmella's BioBrick Cloning Main project page
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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, 1:1000 cDNA dilution; ATF2 insert = 906: ATF2_f1/ ATF2_r1
  3. SKNSH C001, 1:1000 cDNA dilution; same
  4. K562 C001, 1:1000 cDNA dilution; same


Reagent Rxn1 Rxn2 Rxn3 Rxn4 Expected:
1. KAH60 = 6637
2. ATF2 = 906
3. ATF2 = 906
4. ATF2 = 906
Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.0 1.0 1.0
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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