Haynes Lab:Notebook/Jan/2015/04/08

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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
  1. BD004 + MV2 = ~10 colonies
  2. MV2 only (neg) = 0 colonies
  3. BD004 only (neg) = ~15 colonies
    • Possible success: Insert-only had more colonies than insert+vector. Pik 6 colonies for 5 mL cultures/ streak plate. Verify minipreps via restriction digests.



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