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

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03/21/13

  • 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) 03/22/13
1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 lawn
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 lawn
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 lawn
4. MV9(X dp)/ 25 ng  
5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase lawn
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


3/22/13

  • Plates have bacterial lawns. Used plates from Behzad's "LB Plane Agar" sleeve because it was marked with red tape and nobody, conveniently, reported that we ran out of LB amp. Thanks, guys.


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