User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/23

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12/23/09

  • ✓ Minipreps: KAH120-125/MV2 (2 each); KAH135(2)
  • ✓ Assemblies: Strep apt into JDS42 & 43
  • ✓ Cultures: KAH93(2), KAH121-125/MV2(1 each), KAH120/MV2(4)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
MV2 = 4529
1,2. KAH120/MV2 = 7930
3,4. KAH121/MV2 = 8102
5,6. KAH122/MV2 = 8174
7,8. KAH123/MV2 = 8002
9,10. KAH124/MV2 = 8174
11,12. KAH125/MV2 = 8246
13. KAH135 = 2353
File:KAH 122309 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Grow new cultures for low yield plasmids: KAH121-125/MV2 (1 each)
--> Pick more colonies for KAH120/MV2 (4)


Assemblies
--> Continued from 12/22/09

> Ligations

Ligation Plate results (lig : neg crtl) 12/24/09
1. Strep apt(NotI dsoligo)/50, 0.4 ng + JDS42(NotI)/7468, 30 ng Strep apt/JDS42 0 colonies
2. JDS42(NotI)/ 30 ng  
1. Strep apt(NotI dsoligo)/50, 0.4 ng + JDS43(NotI)/7478, 30 ng Strep apt/JDS43 o colonies
2. JDS42(NotI)/ 30 ng  
  1 2 3 4
Insert DNA 0.5 --- 0.5 ---
Vector DNA 6.0 6.0 6.0 6.0
2x lgn buf (Roche) 7.0 7.0 7.0 7.0
T4 ligase (NEB) 0.5 0.5 0.5 0.5
dH2O --- 0.5 --- 0.5
  14μL 14μL 14μL 14μL --> R.T./ 1 hr.; Add 30μL Z-DH5α