User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/08

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12/08/09

  • ✓ Assemblies: KAH109-116

Assemblies

  1. KAH109: (1) KAH87/(E/S)/1053 + (8) NLS-myc-stop(KAH011)/(E/X)/~3300
  2. KAH110: (2) KAH88/(E/S)/1053 + "
  3. KAH111: (3) KAH89/(E/S)/1053 + "
  4. KAH112: (4) KAH90/(E/S)/1053 + "
  5. KAH113: (5) KAH91/(E/S)/2037 + "
  6. KAH114: (6) KAH92/(E/S)/2040 + "
  7. KAH115: (7) KAH74/(E/S)/861 + "
  8. KAH116: mCh/(E/S)/705 ✓ + "

> Digests (Fermentas FD)

Reagent Volume   File:KAH 120809 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. KAH87(E/S) 0.176 2.45 8.8
2. KAH88(E/S) 0.202 2.01 10.1
3. KAH89(E/S) 0.128 2.76 6.4
4. KAH90(E/S) 0.136 2.44 6.8
5. KAH91(E/S) 0.151 2.28 7.5
6. KAH92(E/S) 0.108 2.50 5.4
7. KAH74(E/S) 0.101 3.24 5.1
8. KAH011(E/X) 0.863 2.00 43.2

> Ligations

Ligation Plate results (lig : neg crtl) 12/09/09
1. KAH87(E/S)/1053, 13 ng + KAH011(E/X)/3300, 20 ng KAH109 >10:1 (Pick 2)
2. KAH88(E/S)/1053, 13 ng + " KAH110 >10:1 (Pick 2)
3. KAH89(E/S)/1053, 13 ng + " KAH110 >10:1 (Pick 2)
4. KAH90(E/S)/1053, 13 ng + " KAH110 >10:1 (Pick 2)
5. KAH91(E/S)/2037, 25 ng + " KAH110 >10:1 (Pick 2)
6. KAH92(E/S)/2040, 25 ng + " KAH110 >10:1 (Pick 2)
7. KAH74(E/S)/861, 10 ng + " KAH110 >10:1 (Pick 2)
8. mCh(E/S)/705, 9 ng + " KAH110 >10:1 (Pick 2)
9. KAH011(E/X) 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 1.5 1.3 2.0 1.9 3.3 4.5 2.0 1.1 ---
Vector DNA 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 3.0 3.2 2.5 2.6 1.2 --- 2.5 3.4 4.5
  12μL 12μL 12μL 12μL 12μL 12μL 12μL 12μL 12μL --> R.T./~10 min.; Add 20 μL Z-DH5α