User:Karmella Haynes/Notebook/BioBrick cloning/2012/06/18

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06/18/12

  • ✓ Assemblies: pLac-O2/V0120



Assemblies

  1. p2547 O2/V0120: p2547 O2/(X/P PCR)/~100 + V0120-PEST/(X/P)/~3200
  2. p2547 O2/V0120: p2547 O2/(X/P PCR)/~100 + V0120-VP64/(X/P)/~3200

> Digests (Fermentas FD)
--> Try two new backbone sources, PEST and VP64 basic parts from my plasmid stock

Reagent Volume   Cloning digest
30 μL/lane, 1% agarose
DNA (plasmid) 20.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 5.0
  30 μL --> 37°C/ ~10 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. V0120-PEST (X/P) 0.096 1.917 95.9 --> diluted 1/2 = 48.0
2. V0120-VP64 (X/P) 0.031 1.916 30.8


> Ligations

Ligation Plate results (lig : neg crtl) 06/19/12
1. p2547 O2/(X/P PCR)/~100, 1.6 ng + V0120-PEST(X/P)/~3200, 25 ng p2547 O2/V0120 no colonies
2. V0120-PEST(X/P), 25 ng  
3. p2547 O2/(X/P PCR)/~100, 1.6 ng + V0120-VP64(X/P)/~3200, 25 ng p2547 O2/V0120 no colonies
4. V0120-VP64(X/P), 25 ng  
  1 2 3 4
Insert DNA 0.5 --- 0.5 ---
Vector DNA 0.5 0.5 0.8 0.8
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 3.0 3.5 2.7 3.2
  10 μL 10 μL 10 μL 10 μL

---> Add 30 μL DH5α Turbo; plate on Amp