User:Karmella Haynes/Notebook/BioBrick cloning/2010/06/03

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06/03/10

  • ✓ Assemblies: redo MV8 and MV3



Assemblies

  1. MV8: MV1/(BsaBI/BstBI)/4605 + Hygro PCR/(BsaBI/BstBI)/1023
  2. MV8: MV1/(BsaBI/BstBI)/3919 + "

> Ligations

Ligation Plate results (lig : neg crtl) 06/04/10
1. Hygro PCR(B/B)/1023, 11 ng + MV1(B/B)/4605, 25 ng MV8 1:1
2. MV1(B/B)/ 25 ng ~ 30 colonies
3. Hygro PCR(B/B)/1023, 13 ng + MV7(B/B)/3919, 25 ng MV3 1:1
4. MV7(B/B)/ 25 ng ~ 30 colonies
  1 2 3 4
Insert DNA 1.2 --- 1.5 ---
Vector DNA 0.8 0.8 0.5 0.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 2.0 3.2 2.0 3.5
  10 μL 10 μL 10 μL 10 μL

--> R.T./ 1 hour; Use 25 μL Turbo DH5α; Heat shock and recover for 30 min.

--> Cloning failed. Try Hygro primers with longer extensions to improve cutting.