User:Karmella Haynes/Notebook/BioBrick cloning/2015/01/22: Difference between revisions

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Line 59: Line 59:
| 2x GoTaq green || 12.5 || 12.5 || 12.5 || 12.5  
| 2x GoTaq green || 12.5 || 12.5 || 12.5 || 12.5  
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || --- || --- || --- || ---
|-
| &nbsp; || 25 μL --> 37°C/ ~30 min.
|}
|}
Program:
* 95°C, 3 min
* 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
* 72°C, 3 min
* 4°C ∞




Revision as of 11:58, 22 January 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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 Rxn3 Rxn4
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 --- --- --- ---

Program:

  • 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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