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

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mm/dd/yy

  • Planning: 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 ATF2 insert = 906: ATF2_f1/ ATF2_r1
  3. SKNSH "
  4. K562 "


Reagent Rxn1 Rxn2
Template 1.0 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 ---
  25 μL --> 37°C/ ~30 min.


Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight

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