User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/18

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01/18/10

  • ✓ Cultures: KAH94, 95/JDS49 (not 47!) (6 each); KAH94, 95/JDS48 (6 each)
  • ✓ Minipreps: KAH118/MV2 (4)
  • ✓ Assemblies: KAH139, 140, 141

Minipreps
> Check with E/P digests

Reagent Volume Expected:
MV2 = 4529
1-4. KAH118/MV2 = 7930
File:KAH 011810 gel2.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.

--> None have correct size for MV2 vector. Pick 12 more colonies.


Assemblies

  1. KAH139: HPK-Kstop(KAH103)/(E/S)/546 + (1) KAH137/(E/X)/~5593
  2. KAH140: HPK-K(KAH104)/(E/S)/540 + "
  3. KAH141: HPK-K(KAH104)/(E/S)/540 + (2) KAH138/(E/X)/~5181

> Digests (Fermentas FD)

Reagent Volume   File:KAH 011810 gel1.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. KAH137(E/X) 0.526 1.79 26.3
2. KAH138(E/X) 0.858 1.82 42.9


> Ligations

Ligation Plate results (lig : neg crtl) 01/19/10
1. KAH103(E/S)/546, 6 ng + KAH137(E/X)/5593, 30 ng KAH139 >10:1 (Pick 2)
2. KAH104(E/S)/540, 6 ng + " KAH140 >10:1 (Pick 2)
3. KAH137(E/X)/ 30 ng + "  
4. KAH104(E/S)/540, 17 ng + KAH138(E/X)/1981, 30 ng KAH141 >10:1 (Pick 2)
5. KAH138(E/X)/ 30 ng + "  
  1 2 3 4 5
Insert DNA 0.9 0.9 --- 1.3 ---
Vector DNA 1.1 1.1 1.1 0.7 0.7
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 2.0 2.0 2.9 2.0 3.3
  10μL 10μL 10μL 10μL 10μL --> R.T./ ~15 min.; Add 20 μL Z-DH5α