User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/20

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11/20/09

  • Minipreps: KAH86(2), 87(2), 88(2), 89(2), 90(2), 91(2), 92(2); KAH84(2, re-pick)
  • Digests: KAH77, 78, 79 (E/P/ScaI triple digests)
  • Assemblies: KAH011, 012; KAH93, 94, 95

Minipreps
> Check with E/P digests
> Include KAH77-79 E/P/ScaI digests (ScaI cuts V0120)

Reagent Volume   Expected:
1,2. KAH86 = 2726
3,4. KAH87 = 1053
5,6. KAH88 = 1053
7,8. KAH89 = 1053
9,10. KAH90 = 1053
11,12. KAH91 = 2037
13,14. KAH92 = 2040
15,16. KAH84 = 615
17,18. KAH77 = 3480 (& 1784, 1439)
19-21. KAH78 = 3652 (& 1784, 1439)
22-24. KAH79 = 3724 (& 1784, 1439)
Miniprep check 11/20/09
15μL/lane, 1% gel
  (1-16) (17-24)
DNA(plasmid) 2.0 1.0
10X buffer 1.5 1.5
EcoRI 1.0 1.0
PstI 1.0 1.0
ScaI --- 1.0
dH2O 9.5 15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH011: NLS-myc-stop/(X/P ds-oligo)/64 ✓ + (1) V0120/(X/P)/3200
  2. KAH012: Kstop/(X/P ds-oligo)/24 ✓ + "
  3. KAH93: (2) 2xHS4-Ubc-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH77)/(E/S)/3480 + (5) 5xLuc target-polyA-2xHS4 (KAH67)/(E/X)/4553
  4. KAH94: (3) 2xHS4-HPK-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH78)/(E/S)/3652 + "
  5. KAH95: (4) 2xHS4-CMV-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH79)/(E/S)/3724 + "

> Digests (Fermentas FD)
--> Triple digest KAH977-79 to cut V0120

Reagent Volume   Isolate:
1. V0120 = 2726
2. KAH77 = 3480
3. KAH78 = 3652
4. KAH79 = 3724
5. KAH67 = 4553
Cloning digest 11/20/09
30 μL/lane, 1% gel
  (1,5) (2-4)
DNA (plasmid) up to 25 μL 5
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ScaI --- 1.0
dH2O --- 19
  30 μL 30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. V0120(X/P) 0.097 2.87 4.83
2. KAH77(E/S) 0.405 2.13 20.24
3. KAH78(E/S) 0.432 2.07 21.62
4. KAH79(E/S) 0.396 2.06 19.79
5. KAH67(E/X) 1.885 1.87 94.25


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH011(X/P)/64, > 0.8 ng + V0120(X/P)/3200, 20 ng KAH011 10:1 (Pick 2)
2. KAH012(X/P)/24, > 0.3 ng + V0120(X/P)/3200, 20 ng KAH012 >10:1 (Pick 2)
3. V0120(X/P) 20 ng  
4. KAH77(E/S)/3480, > 46 ng + KAH67(E/X)/4553, 30 ng KAH93 3:1 (Pick 2)
5. KAH78(E/S)/3652, > 48 ng + " KAH94 5:1 (Pick 2)
6. KAH79(E/S)/3724, > 49 ng + " KAH95 5:1 (Pick 2)
7. KAH67(E/X) 30 ng
  1 2 3 4 5 6 7
Insert DNA 0.5 0.5 --- 2.5 2.5 2.5 ---
Vector DNA 4.0 4.0 4.0 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 5.5 5.5 5.5 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
dH2O --- --- 0.5 1.0 1.0 1.0 3.5
  11 μL 11 μL 11 μL 10 μL 10 μL 10 μL 10 μL --> R.T./ 10 min.; Add 25 μL Z-DH5α