User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/01

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12/01/09

  • ✓ Cultures: KAH011(8)
  • ✓ Assemblies: KAH93, 94, 95 into pcDNA3.1+puro/ΔpCMV(MV2), use E/P; KAH105, 106, 107, 108

Assemblies

  1. KAH93/MV2: (1) KAH93/(E/P)/4839 + (4) pcDNA3.1+puro/ΔCMV(MV2)/(E/P)/4529
  2. KAH94/MV2: (2) KAH94/(E/P)/5011 + "
  3. KAH95/MV2: (3) KAH95/(E/P)/5083 + "
  4. KAH105: (5) Ubc-Kstop(KAH102)/(E/S)/374 + Gal4DB-PEST-2xmCh-NLS-stop(KAH68)/(E/X)/5614 ✓
  5. KAH106: (6) Ubc-K(BART19)/(E/S)/368 + "
  6. KAH107: (7) HPK-Kstop(KAH103)/(E/S)/546 + "
  7. KAH108: (8) HPK-K(KAH104)/(E/S)/540 + "

> Digests (Fermentas FD)

Reagent Volume   File:KAH 120109 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. KAH93(E/P) 0.681 1.83 34.07
2. KAH94(E/P) 0.387 1.99 19.36
3. KAH95(E/P) 0.221 1.73 11.04
4. MV2(E/P) 1.126 1.87 56.28
5. KAH102(E/S) 0.382 1.89 19.08
6. BART19(E/S) 0.172 2.89 8.60
7. KAH103(E/S) 0.524 1.96 26.21
8. KAH104(E/S) 0.517 1.90 25.83

> Ligations

Ligation Plate results (lig : neg crtl) 12/02/09
1. KAH93(E/P)/4839, 43 ng + MV2(E/P)/4529, 20 ng KAH93/MV2 1:1 (re-do)
2. KAH94(E/P)/5011, 44 ng + " KAH94/MV2 1:1 (re-do)
3. KAH95(E/P)/5083, 45 ng + " KAH95/MV2 1:1 (re-do)
4. MV2(E/P)/ 20 ng (>100 colonies)
5. KAH102(E/S)/374, 3 ng + KAH68(E/X)/5614, 20 ng KAH105 10:1 (Pick 2)
6. BART19(E/S)/368, 3 ng + " KAH106 10:1 (Pick 2)
7. KAH103(E/S)/546, 4 ng + " KAH107 10:1 (Pick 2)
8. KAH104(E/S)/540, 4 ng + " KAH108 10:1 (Pick 2)
9. KAH68(E/X)/ 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 1.3 2.3 4.1 --- 0.5 0.5 0.5 0.5 ---
Vector DNA 0.4 0.4 0.4 0.4 0.5 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 5.5 5.5 5.5 5.5 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
dH2O 2.8 1.8 --- 4.1 3.0 3.0 3.0 3.0 3.5
  11μL 11μL 11μL 11μL 10μL 10μL 10μL 10μL 10μL --> R.T./ ~10 min.; Add 25 μL Z-DH5α