Haynes Lab:Notebook/Jan/2015/04/08: Difference between revisions

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* BD004: insert BD004(E/S)/4118 + vector MV2(E/X)/4529
* BD004: insert BD004(E/S)/4118 + vector MV2(E/X)/4529
* Use Roche 2x buffer and NEB T4 ligase
* Use Roche 2x buffer and NEB T4 ligase
* Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/03/27
* Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/04/07




Revision as of 11:33, 8 April 2015

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04/08/15

  • Assembly - BD004/MV2


Assembly


  • Calculations
    • Vector - MV2(E/X); 50 ng = ## μL
    • Insert - BD004(E/S); (4096/4529)*2*50 = 90.4 ng = ### μL


  • Ligations
  1. BD004 + MV2
  2. MV2 only (neg)


Reagent 1 2
DNA insert 1.4 ---
DNA vector 7.7 7.7
2x buffer 9.6 9.6
T4 ligase 0.5 0.5
dH2O --- 1.4
  19.2 μL 19.2 μL

--> RT/ ~30 min.

  • Add 50 μL DH5-α turbo
  • 5 min./ ice
  • Plate on 100 ug/mL amp agar. Grow at 37 °C o/n


RESULTS

  • After ~7 hours of growth, saw tiny colonies only on ligation plate
  • To save time, picked two colonies from ligation plate
    • Make streaks on fresh agar
    • Used tips to inoculate 5 mL cultures


  • 4/07/15
    • Success! ~20 colonies in ligation plate, zero on negative ctrl.
    • Growth on streak plate and in cultures



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