User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/03

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12/03/09

  • ✓ Minipreps: KAH105(2), 106(2), 107(2), 108(2)
  • ✓ Cultures: KAH93/MV2(2), 94/MV2(2), 95/MV2(2)
  • ✓ Assemblies: KAH99, 100, 101, 105, 106, 107, 108 into MV2

Minipreps
> Check with E/P/ScaI triple digests

Reagent Volume Expected:
1,2. KAH105 = 2794
3,4. KAH106 = 2788
5,6. KAH107 = 2966
7,8. KAH108 = 2960
File:KAH 120309 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 4.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
ScaI 1.0
dH2O 6.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH99/MV2: (1) KAH99/(E/P)/2913 + MV2/(E/P)/4529 ✓
  2. KAH100/MV2: (2) KAH100/(E/P)/3085 + "
  3. KAH101/MV2: (3) KAH101/(E/P)/3157 + "
  4. KAH105/MV2: (4) KAH105/(E/P)/2794 + "
  5. KAH106/MV2: (5) KAH106/(E/P)/2788 + "
  6. KAH107/MV2: (6) KAH107/(E/P)/2966 + "
  7. KAH108/MV2: (7) KAH108/(E/P)/2960 + "

> Digests (Fermentas FD)
--> E/P/ApaLI for KAH99-101
--> E/P/ScaI for KAH105-108

Reagent Volume   File:KAH 120309 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 24 μL
10x buffer 3.0
EcoRI 1.0
PstI 1.0
enzyme 3 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH99(E/P) 0.405 1.99 20.27
2. KAH100(E/P) 0.429 1.93 21.43
3. KAH101(E/P) 0.398 2.09 19.90
4. KAH105(E/P) 0.198 1.92 9.88
5. KAH106(E/P) 0.240 2.18 11.98
6. KAH107(E/P) 0.225 1.99 11.23
7. KAH108(E/P) 0.221 2.58 11.06


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH99(E/P)/2913, 32 ng + MV2(E/P)/4529, 25 ng KAH99/MV2 10:1 (Pick 2)
2. KAH100(E/P)/3085, 34 ng + " KAH100/MV2 10:1 (Pick 2)
3. KAH101(E/P)/3157, 35 ng + " KAH101/MV2 10:1 (Pick 2)
4. KAH105(E/P)/2794, 31 ng + " KAH105/MV2 10:1 (Pick 2)
5. KAH106(E/P)/2788, 31 ng + " KAH106/MV2 10:1 (Pick 2)
6. KAH107(E/P)/2966, 33 ng + " KAH107/MV2 10:1 (Pick 2)
7. KAH108(E/P)/2960, 33 ng + " KAH108/MV2 10:1 (Pick 2)
8. MV2(E/P)/ 25 ng  
  1 2 3 4 5 6 7 8
Insert DNA 1.6 1.6 1.8 3.0 2.6 3.0 3.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 1.4 1.4 1.2 --- 0.4 --- --- 3.0
  10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL --> R.T./ 10 min.; Add 25 μL Z-DH5α