User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/26

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01/26/11

  • ✓ Minipreps: SP1AB 3xmut (24)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-24. SP1AB 3x mut
w.t. = 259, 317, 454, 587
site 1 = 454, 576, 587
site 2 = 317, 454, 846
site 3 = 259, 317, 1041
sites 1/2 = 454, 1163
sites 1/3 = 576, 1041
sites 2/3 = 317, 1300
sites 1/2/3 = 1617
Minipreps E/P digest 01/26/11
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.

--> Conclusions: Site #1 was not mutated at all. Keep clones 1-6 to confirm no off-target mutations, then use one good clone to mutate site#1. Order 2 more mut_SP1A primers




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