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

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'''RESULTS'''
'''RESULTS'''
* After ~7 hours of growth, saw tiny colonies only on ligation plate
* 4/09/15
* To save time, picked two colonies from ligation plate
** tba
** 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




Revision as of 16:58, 8 April 2015

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04/08/15

  • Assembly - BD004/MV2


Assembly


  • Calculations
    • Vector - MV2(E/X); 50 ng = 1.5 μL
    • Insert - BD004(E/S); (4096/4529)*2*50 = 90.4 ng = 2.8 μL


  • Ligations
  1. BD004 + MV2
  2. MV2 only (neg)
  3. BD004 only (neg)- to check if any of old vector co-purified w/ insert; bands were very close on gel


Reagent 1 2 3
DNA insert 2.8 --- 2.8
DNA vector 1.5 1.5 ---
2x buffer 5.0 5.0 5.0
T4 ligase 0.5 0.5 0.5
dH2O 0.2 3.0 1.7
  10.0 μL 10.0 μL 10.0 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

  • 4/09/15
    • tba



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