User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/27

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11/27/09

  • Assemblies: KAH99, 100, 101, 102, 103, 104

Assemblies

  1. KAH99: (1) Ubc-5xGal4 (KAH83)/(S/P)/~3643 + (4) K-PEST-2xAmCyan-NLS-stop-polyA(KAH82)/(X/P)/2464
  2. KAH100: (2) HPK-5xGal4 (KAH84)/(S/P)/~3815 + "
  3. KAH101: (3) CMV-5xGal4 (KAH85)/(S/P)/~3887 + "
  4. KAH102: Ubc/(E/S)/344 ✓ + (5) Kstop(KAH012)/(E/X)/~3200
  5. KAH103: HPK/(E/S)/516 ✓ + "
  6. KAH104: HPK/(E/S)/516 ✓ + K/(E/X)/~3200 ✓

> Digests (Fermentas FD)

Reagent Volume   File:KAH 112709 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. KAH83 (S/P) 0.420 1.80 21.01
2. KAH84 (S/P) 0.213 1.62 10.67
3. KAH85 (S/P) 0.466 1.83 23.30
4. KAH82 (X/P) 0.645 1.82 32.24
5. KAH012 (E/X) 0.278 1.80 13.92


> Ligations

Ligation Plate results (lig : neg crtl) 11/29/09
1. KAH82(X/P)/2464, >41 ng + KAH83(S/P)/3643, 30 ng KAH99 >10:1 (Pick 2)
2. KAH83(S/P)/ 30 ng  
3. KAH82(X/P)/2464, >39 ng + KAH84(S/P)/3815, 30 ng KAH100 >10:1 (Pick 2)
4. KAH84(S/P)/ 30 ng  
5. KAH82(X/P)/2464, >38 ng + KAH85(S/P)/3887, 30 ng KAH101 >10:1 (Pick 2)
6. KAH85(S/P)/ 30 ng  
7. Ubc(E/S)/344, >4 ng + Kstop(E/X)/3200, 20 ng KAH102 >10:1 (Pick 2)
8. HPK(E/S)/516, >6 ng + " KAH103 >10:1 (Pick 2)
9. Kstop(E/X)/ 20 ng  
10.HPK(E/S)/516, >6 ng + K(E/X)/3200, 20 ng KAH104 >10:1 (Pick 2)
11. K(E/X)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10 11
Insert DNA 1.3 --- 1.2 --- 1.2 --- 0.4 0.6 --- 0.6 ---
Vector DNA 1.4 1.4 2.8 2.8 1.3 1.3 1.4 1.4 1.4 0.6 0.6
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 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 1.0 1.0 1.0 1.0 1.0
dH2O 1.3 2.6 --- 1.2 1.5 2.7 2.2 2.0 2.6 2.8 3.4
  10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL ---> R.T./ ~10 min.; Add 18 μL Z-DH5α