User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/11

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04/11/13

  • Oligo assembly for new mammalian vector MV9
  • Chem. Competent cell prep: DH5α Turbo (from NEB); 5 mL culture/ ~8 hours; 2x 250 mL cultures o/n



Assemblies

  • Try again with the old boil and cool method...
    • Aluminum foil lid on the beaker to prevent condensation
    • Different amounts of 100 μM oligos


  • 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. Cover the beaker with aluminum foil to keep the air above the tube warm.
    • Turn off the heat source and allow the aluminum foil-covered water bath to slowly cool to room temperature (25°C) for several hours.
  1 2 3 4
100 μM Oligo 1 3.0 2.0 1.0 0.5
100 μM Oligo 2 3.0 2.0 1.0 0.5
100 μM Oligo 3 3.0 2.0 1.0 0.5
100 μM Oligo 4 3.0 2.0 1.0 0.5
10x annealing buffer 2.0 2.0 2.0 2.0
dH2O 6.0 10.0 14.0 16.0
  20 μL 20 μL 20 μL 20 μL


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


  • Ligations
    • Inserts have no 5'-phosphates, so dp vector will not work! Switch to non-dp vector. expect high bg on neg ctrl. Will need to screen ligations.
Ligation Plate results (lig : neg crtl) 03/22/13
1. 1 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng 20 colonies
2. 2 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng 20 colonies
3. 3 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng 20 colonies
4. 3 (ds oligo S/S)/76, 0.5 μL + MV2(X)/4529, 25 ng 20 colonies
5. MV2(X dp)/ 25 ng ~100 colonies


  1 2 3 4 5
Insert DNA 0.5 0.5 0.5 0.5 ---
Vector DNA 0.3 0.3 0.3 0.3 0.3
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 3.2 3.2 3.2 3.2 3.7
  10 μL 10 μL 10 μL 10 μL 10 μL
  • Incubate ligation for 30 min./ room temp
  • Pre-warm agar-amp plates with lids closed
  • Add 30 μL DH5α (fresh from NEB); 2 min/ ice
  • Heat shock at 42°C, 30 sec. Place immediately on ice.
  • Add 100 μL plain LB to cells.
  • Plate on 100 μg/mL amp; incubate at 37°C


4/12/13

  • More colonies on the neg. ctrl. than the insert + vector plates.
  • Do colony PCR using MV2 primers; pick 8 colonies from each plate 1 - 4; also do PCR on 4 colonies from the neg. ctrl.



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