User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/02

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04/02/10

  • ✓ Minipreps: KAH014 (8); KAH96 (4)
  • ✓ Sequencing order: KAH014, 96



Minipreps
> Check KAH014 with E/P digests; include DPRE w.t. E/P, E/S
> Check KAH96 with E/P digests; include KAH86

Reagent Volume Expected:
1-8. KAH014 = 1740
9. DPRE(E/P) = 1001, 662, 77
10. DPRE(E/S) = 1740
11-14. KAH96 = 2825
KAH86 = 2726
File:KAH 040210 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
enzyme 2 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.



Sequencing (Genewiz, standard)

  1. KAH014-3 P0001: bad (w.t. PstI #1)
  2. KAH014-3 P0002: good (mut PstI #2)
  3. KAH014-4 P0001: good (mut PstI #1); single bp variation @ 441
  4. KAH014-4 P0002: good (mut PstI #2)
  5. KAH96-1 P0001: low quality sequence (non-specific)
  6. KAH96-1 P0002: good
  7. KAH96-2 P0001: good
  8. KAH96-2 P0002: good

--> Keep KAH014-4 and KAH96-2