User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20

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03/20/13

  • Oligo annealing, round 2
  • Assembly: annealed part into MV2


Assemblies

  • New strategy for mammalian vector:
    • Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter. This way, the vector can be customized to carry different promoters.
    • Insertion of the ds oligo should destroy the vector's XbaI site
    • Will need to check inserts for copy number and orientation


  • Oligo annealing
  • Redo oligo annealing with 99.9 and 97.9°C starting temperatures...
  • Previous Thermal cycling (3/19/13)
    • 99.9, 10 min.
    • [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35
    • 25°C, ∞
    • Labeled tubes A1, A2; Stored on bench o/n
  • New Thermal cycling (3/20/13)
    • 99.9, 10 min.
    • [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36
    • 25°C, ∞
    • Labeled tubes A3, A4


  • Digest (Fermentas FD)
    • MV2, XbaI only
Reagent Volume
DNA (plasmid) 20.0 μL
10x buffer 3.0
XbaI 2.0
dH2O 5.0
  30 μL --> 37°C/ ~15 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. MV2 (XbaI) 0.081 1.841 81.3


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) 6.2 μL (500 ng)
10x buffer d.p. 2.0
rapid alk. phosphatase 1.0
dH2O 10.8
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) 03/21/13
1. A1 (ds oligo S/S)/size, 0.5 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
2. A1 (ds oligo S/S)/size, 1.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
3. A1 (ds oligo S/S)/size, 2.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
4. MV9(X dp)/ 25 ng  
5. A3 (ds oligo S/S)/size, 0.5 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
6. A3 (ds oligo S/S)/size, 1.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
7. A3 (ds oligo S/S)/size, 2.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
8. MV9(X dp)/ 25 ng  
  1 2
Insert DNA 0.5 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  10 μL 10 μL



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