Haynes Lab:Notebook/Jan/2015/04/06

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04/06/15

  • Assembly - DPRE into "BD004"
  • Sequencing - BD006/MV2



Assembly


  • Calculations
    • Vector - delBD004(E/X); 50 ng = 7.7 μL
    • Insert - DPRE(E/S); (1740/7337)*2*50 = 23.7 ng = 1.4 μL


  • Ligations
  1. DPRE + delBD004
  2. delBD004 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



Sequencing: BD006/MV2

Sample # Template Primer Benchling ref file Results (04/10/15)
1. BD006MV2_1 DD122B ChromatinSensor1_MV2 Good qual, t/C conflict at 605
2. BD006MV2_1 BGHR " Bad qual
3. BD006MV2_2 DD122B " Good qual, t/C conflict at 605
4. BD006MV2_2 BGHR " Good qual
5. BD006MV2_3 DD122B " Good qual, t/C conflict at 605
6. BD006MV2_3 BGHR " Good qual
7. BD006MV2_4 DD122B " Good qual, t/C conflict at 605
8. BD006MV2_4 BGHR " Good qual



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