User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/01

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04/01/10

  • ✓ Cultures: KAH96 rebuilt (4); KAH014 (8)
  • ✓ Minipreps: KAH014(DPRE) (4)
  • ✓ Cultures: KAH014 (8)
  • ✓ Sequencing: KAH014; KAH144, 145 (confirmation of EED expression cassettes)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. KAH014 = 1740
Double mut = 1740
Mut 1st Pst = 1078, 662
Mut 2nd Pst = 1663, 77
5. DPRE (no mut) = 1001, 662, 77
E/P digest 4/01/10
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.

--> KAH014-1 and 2 might be good (77 bp band?). Send for sequencing.
--> Pick 8 more clones from colony plate.


Sequencing (Genewiz, standard)

  1. KAH014-1 P0001: bad (w.t. PstI #1)
  2. KAH014-1 P0002: good (mut PstI #2)
  3. KAH014-2 P0001: bad (w.t. PstI #1)
  4. KAH014-2 P0002: good (mut PstI #2)
  5. KAH144 P0001: good
  6. KAH144 P0002: good
  7. KAH145 P0001: good
  8. KAH145 P0002: good



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