User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/21

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mm/dd/yy

  • Assemblies: retry MV9
  • T4 ligase quality check: Behzad's tube



Assemblies

  • Try again, but with the old boil and cool method


  • Oligo annealing
  • See 03/19/13 for details about the oligos and reaction set-up
    • Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).
    • Float the oligo mixtures in the boiling water for 10 min. If there is condensation, carefully & quickly remove the tube and shoe the liquid down to the bottom and put the tubes back into the water.
    • Turn off the heat source and allow the water bath to slowly cool to room temperature (25°C) for several hours.


  • Digest (Fermentas FD) - MV2 vector, 03/20/13
  • Measure conc. - 03/20/13
  • Dephosphorylation (Roche) - MV2 XbaI-cut vector, 03/20/13


  • Ligations
Ligation Plate results (lig : neg crtl) 03/21/13
1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
4. MV9(X dp)/ 25 ng  
5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase ...
6. MV9(X no dp)/ 25 ng  
  1 2 3 4 9 10
Insert DNA 0.5 1.0 2.0 --- --- ---
Vector DNA 1.0 1.0 1.0 1.0 0.3 0.3
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 ---
dH2O 2.5 2.0 1.0 3.0 3.7 4.7
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL
  • Pre-warm agar-amp plates with lids closed
  • 30 min./ room temp
  • Add 30 μL DH5α; 5 min/ ice
  • Plate on 100 μg/mL amp; incubate at 37°C


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