User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/24

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03/24/10

  • ✓ Minipreps: KAH143 (4)
  • ✓ Assembly: KAH144
  • ✓ Sequencing order: KAH86, 96, 143

Minipreps
> Check with E/P digest

Reagent Volume Expected:
1-4. KAH143 = 1853
File:KAH 032410 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.

Assemblies

  1. KAH144: KAH104/(E/S)/540 ✓ + (1) KAH142/(E/X)/~5053

> Digests (Fermentas FD)

Reagent Volume   File:KAH 032410 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) 5.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH143 (E/X) 0.125 1.97 6.3

--> KAH104(E/S) = 25.8 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 03/25/10
1. KAH104(E/S)/540, 5.3 ng + KAH143(E/X)/~5053, 25 ng new BioBrick 5:1 (Pick 4)
2. KAH143(E/X)/ 25 ng ~100 colonies
  1 2
Insert DNA 0.5 ---
Vector DNA 4.0 4.0
2x lgn buf (Roche) 5.5 5.5
T4 ligase (NEB) 1.0 1.0
dH2O --- 0.5
  11 μL 11 μL

--> R.T./ ~10 min., Add to 50 μL Z-DH5α


Sequencing order (same day, placed @ 8pm today, results Friday)

  1. KAH86 P0001: missing HSVtkTATA-K promoter
  2. KAH86 P0002: good
  3. KAH96 P0001: missing HSVtkTATA-K promoter
  4. KAH96 P0002: good
  5. KAH143 P0001: good
  6. KAH143 (check NLSstop) P0002: good (NLS stop2 confirmed)