User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/26

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03/26/10

  • ✓ Minipreps: KAH144 (4)
  • ✓ Assembly: KAH145

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. KAH144 = 2399
Hover name
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.

Assemblies

  1. KAH145: (1) KAH144/(S/P)/~5599 + KAH51/(X/P)/1160 ✓

> Digests (Fermentas FD)

Reagent Volume   Hover name
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 15.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH144 (S/P) 0.217 1.91 10.8

--> KAH51 (X/P) = 42.9


> Ligations

Ligation Plate results (lig : neg crtl) 03/28/10
1. KAH51(X/P)/1160, 10 ng + KAH144(S/P)/~5599, 25 ng KAH145 >10:1 (Pick 4)
2. KAH144(S/P)/ 25 ng ~ 20 colonies
  1 2
Insert DNA 0.5 ---
Vector DNA 2.5 2.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.0 1.5
  10 μL 10 μL

--> R.T./ ~10 min.; Add 50 μL Z-DH5α


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