User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/24

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11/24/09

  • ✓ Minipreps: KAH011(2), 012(2), 93(2), 94(2), 95(2)
  • ✓ Sequencing order (same day): KAH011, 012
  • ✓ Assemblies: KAH96, 97, 98, 99, 100, 101
  • ✓ Cultures: Re-pick KAH94(4), 95(4)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH93 = 4839
3,4. KAH94 = 5011
5,6. KAH95 = 5083
E/P Digest 11/24/09
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH96: 5xGal4/(S/P)/~3300 ✓ + (1) HSVtkTATA-K-2xYFP-NLSstop2-polyA-2xHS4(KAH86)/(X/P/ScaI)/2726
  2. KAH97: (2) 5xGal4-Intron(KAH42)/(S/P)/~4875 + "
  3. KAH98: (3) 5xGal4-2xIntron(KAH47)/(S/P)/~6457 + "
  4. KAH99: (4) Ubc-5xGal4 (KAH83)/(E/S)/443 + (7) K-PEST-2xAmCyan-NLS-stop-polyA(KAH82)/(E/X)/~5664
  5. KAH100: (5) HPK-5xGal4 (KAH84)/(E/S)/615 + "
  6. KAH101: (6) CMV-5xGal4 (KAH85)/(E/S)/687 + "

> Digests (Fermentas FD)
--> Use triple digest for KAH86

Reagent Volume   Cloning gel 11/24/09
30 μL/lane, 1% gel
  (2-7) (1)
DNA (plasmid) ^ to 25μL 15
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ScaI --- 1.0
dH2O --- 9.0
  30μL 30μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH86(X/P) 1.112 1.85 55.62
2. KAH42(S/P) 0.282 1.99 14.08
3. KAH47(S/P) 0.486 1.99 24.28
4. KAH83(E/S) 0.185 2.07 9.27
5. KAH84(E/S) 0.397 1.98 19.85
6. KAH85(E/S) 0.212 2.15 10.58
7. KAH82(E/X) 1.909 1.85 95.43


Ligations Plate results (lig : neg crtl) 11/25/09
1. KAH86(X/P)/2726, >33 ng + 5xGal4(S/P)/3300, 20 ng KAH96 >10:1 (Pick2)
2. 5xGal4(S/P)/ 20 ng  
3. KAH86(X/P)/2726, >34 ng + KAH42(S/P)/4875, 30 ng KAH97 >10:1 (Pick2)
4. KAH42(S/P)/ 30 ng  
5. KAH86(X/P)/2726, >34 ng + KAH47(S/P)/6457, 40 ng KAH98 10:1 (Pick2)
6. KAH47(S/P)/ 40 ng  
7. KAH83(E/S)/443, >6 ng + KAH82(E/X)/5664, 40 ng KAH99, 0 colonies
8. KAH84(E/S)/615, >9 ng + " KAH100, 0 colonies
9. KAH85(E/S)/687, >9 ng + " KAH101, 0 colonies
10. KAH82(E/X)/ 40 ng (0 colonies)
  1 2 3 4 5 6 7 8 9 10
Insert DNA 0.6 --- 0.6 --- 0.6 --- 0.6 0.5 0.9 ---
Vector DNA 0.4 0.4 2.0 2.0 1.6 1.6 0.4 0.4 0.4 0.4
2x lgn buf (Roche) 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
dH2O 3.0 4.0 1.4 2.0 1.8 2.4 3.0 3.1 2.7 3.6
  10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL ---> R.T./ ~10 min.; Add 20 μL Z-DH5α



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