User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/12

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01/12/10

  • ✓ Minipreps: KAH117(4), 118(1), 119/MV2(1)
  • ✓ Cultures: re-pick KAH117/MV2 (4)
  • ✓ Assemblies: KAH117, 118/MV2
  • ✓ Retransform: KAH94, 95



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. KAH117/MV2 = 7758
5. KAH118/MV2 = 7930
6. KAH119/MV2 = 8002
File:KAH 011210 gel2.tif
15 μL/lane; 1% agarose
DNA(plasmid) 5.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 6.5
  15 μL --> 37°C/ ~15 min.

--> Pick 4 remaining colonies from KAH117/MV2 plate
--> Re-assemble KAH117, 118/MV2


Assemblies

  1. KAH117/MV2: KAH117/(E/P)/7758 ✓ + MV2/(E/P)/4529 ✓
  2. KAH118/MV2: KAH118/(E/P)/7930 ✓ + "


> Ligations

Ligation Plate results (lig : neg crtl) 01/13/10
1. KAH117(E/P)/7758, 34 ng + MV2(E/P)/4529, 10 ng KAH117/MV2 16 colonies (Pick 6)
2. KAH118(E/P)/7930, 34 ng + " KAH118/MV2 14 colonies (Pick 6)
3. MV2(E/P)/ 10 ng 16 colonies
  1 2 3
Insert DNA 11.1 11.1 ---
Vector DNA 0.4 0.4 0.4
2x lgn buf (Roche) 12.5 12.5 12.5
T4 ligase (NEB) 1.0 1.0 1.0
dH2O --- --- 11.6
  25μL 24μL 24μL --> R.T./ 30 min.; Add 50μL Z-DH5α

> Digests (Fermentas FD)
--> For cloning KAH94, 95 into new JDS luciferase vectors (miR project)

  1. JDS47/(BglII)/7509
  2. JDS48/(BglII)/7519
Reagent Volume   File:KAH 011210 gel1.tif
30 μL/lane, 1% agarose
DNA (plasmid) 25.0
10x buffer 3.0
BglII 2.0
dH2O ---
  30 μL --> 37°C/ ~1 hr


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. JDS47(BglII) --- --- ---
2. JDS48(BglII) --- --- ---