User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/19

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11/19/09

  • ✓ Minipreps: KAH77(2), 78(3), 79(3), 83(2), 84(2), 85(2)
  • ✓ Assemblies: NLS myc stop (KAH011), K-stop (KAH012); Postpone ---> KAH93, 94, 95
  • ✓ Cultures: KAH86(2), 87(2), 88(2), 89(2), 90(2), 91(2), 92(2); KAH84(2, re-pick); 5 ml ea.

Minipreps
> Check with E/P digests (Fermentas FD)

Reagent Volume Expected:
1,2. KAH77 = 3480
3-5. KAH78 = 3652
6,7. KAH79 = 3724
8,9. KAH83 = 443
10,11. KAH84 = 615
12,13. KAH85 = 687
E/P Digest1 11/19/09
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Note: Re-do KAH77-79 digests tomorrow, triple digest to cut V0120


Assemblies

  1. KAH011: NLS-myc-stop/(X/P ds-oligo)/64 + V0120/(X/P)/3200
  2. KAH012: Kstop/(X/P ds-oligo)/24 + V0120/(X/P)/3200

(tomorow)

  1. KAH93: 2xHS4-Ubc-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH77)/(E/S)/3480 + 5xLuc target-polyA-2xHS4 (KAH67)/(E/X)/4553
  2. KAH94: 2xHS4-HPK-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH78)/(E/S)/3652 + "
  3. KAH95: 2xHS4-CMV-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH79)/(E/S)/3724 + "

> Ligations

Ligation Plate results (lig : neg crtl) 11/20/09
1. NLS-myc-stop(X/P ds-oligo)/64, > 0.8 ng + V0120(X/P)/3200, 20 ng KAH011, 0 colonies (re-do)
2. Kstop(X/P ds-oligo)/24, > 0.3 ng + V0120(X/P)/3200, 20 ng KAH012, 1 colony (re-do)
3. V0120/(X/P)/ 20 ng 0 colonies
  1 2 3
Insert DNA 0.2 0.2 ---
Vector DNA (from Mara) 2.5 2.5 2.5
2X lgn buf (Roche) 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O 1.3 1.3 1.5
  10 μL 10 μL 10 μL --> R.T./ ~15 min.; Add 25μL Zymo DH5α