User:Karmella Haynes/Notebook/BioBrick cloning/2010/06/01

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06/01/10

  • ✓ Assemblies: make MV8 and MV3



Assemblies

  1. MV8: MV1/(BsaBI/BstBI)/4605 + Hygro PCR/(BsaBI/BstBI)/1023
  2. MV3: MV7/(BsaBI/BstBI)/3919 + "

> Digests (NEB)
--> Specific notes

Reagent Volume   Cloning digest 06/02/10
30 μL/lane, 1% agarose
DNA (plasmid) 15.0
10x buffer 4 3.0
BsaBI 1.0
BstBI 1.0
dH2O 10.0
 

--> 60°C/ 1 hr.; 65°C/ 1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. MV1 (BsaBI/BstBI) 0.636 1.80 31.8
2. MV7 (BsaBI/BstBI) 1.061 1.77 53.0


> Ligations

Ligation Plate results (lig : neg crtl) 06/03/10
1. Hygro PCR(B/B)/1023, 11 ng + MV1(B/B)/4605, 25 ng MV8 1:1 (redo)
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 (redo)
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./ ~10 min.; Add 50 μL Turbo DH5α


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