User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/25

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

05/25/11

  • ✓ Cultures/minipreps/digests: KAH184(12)
  • ✓ Assemblies: KAH176fwd/183, 176fwd/184, 177fwd/183, 177fwd/184



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-12. KAH184 = 2191
File:KAH 052511 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. KAH176fwd/183: (1) KAH183/(E/S)/2383 + (3) KAH176/MV8 /(E/X)/7785
  2. KAH176fwd/184: (2) KAH184/(E/S)/2191 + "
  3. KAH177fwd/183: 5' part/(a/b)/2383 + (4) KAH177/MV8 /(E/X)/7978
  4. KAH177fwd/184: 5' part/(a/b)/2191 + "

> Digests (Fermentas FD)

Reagent Volume   File:KAH 052511 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH183 (E/S) 0.258 2.21 12.9
2. KAH184 (E/S) 0.225 1.84 11.2
3. KAH176 (E/X) 0.225 2.20 11.2
4. KAH177 (E/X) 0.393 2.12 19.7


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH183(E/S)/2383, 18 ng + KAH176(E/X)/7785, 30 ng KAH183/176 2:1 (Pick 6)
2. KAH184(E/S)/2191, 16 ng + " KAH184/176 2:1 (Pick 6)
3. KAH176(E/X)/ 30 ng  
4. KAH183(E/S)/2383, 18 ng + KAH177(E/X)/7978, 30 ng KAH183/177 3:1 (Pick 6)
5. KAH184(E/S)/2191, 16 ng + " KAH184/177 3:1 (Pick 6)
6. KAH177(E/X)/ 30 ng  
  1 2 3 4 5 6
Insert DNA 1.5 1.5 --- 2.5 2.5 ---
Vector DNA 2.5 2.5 2.5 1.5 1.5 1.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- 1.5 --- --- 2.5
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


--> Add 10 μL to 30 μL DH5α-Turbo