User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/18

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11/18/09

  • ✓ Cultures: KAH77(2), 78(3), 79(3), 83(2), 84(2), 85(2)
  • ✓ Assemblies: KAH86, 87, 88, 89, 90, 91, 92
  • ✓ Oligo annealing: NLS myc stop (KAH011), K-stop (KAH012), BglII adapters

Assemblies

  1. KAH86: HSVtkTATA-K (KAH48)/(S/P)/~3200 ✓ + (1) 2xYFP-NLSstop2-polyA-2xHS4 (KAH81)/(X/P)/2640
  2. KAH87: (2) huPcCD/(E/S)/186+3200 + (8) mCh-VP64 (KAH74)/(E/X)/4061
  3. KAH88: (3) mPcCD/(E/S)/186+3200 + "
  4. KAH89: (4) flyPcCD/(E/S)/186+3200 + "
  5. KAH90: (5) fshPcCD/(E/S)/186+3200 + "
  6. KAH91: (6) hCBX8/(E/S)/1170 + "
  7. KAH92: (7) flyPc/(E/S)/1173 + "

> Digests (Fermentas FD)
--> Gel purify 1, 6, 7, 8
--> Zymo clean & conc. 2-5 (for dephos.)

Reagent Volume
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. KAH81 (X/P) 0.519 1.67 25.9
2. huPcCD (E/S) 1.590 1.76 79.5
3. mPcCD (E/S) 1.604 1.76 79.5
4. flyPcCD (E/S) 0.903 1.66 45.2
5. fshPcCD (E/S) 1.482 1.74 74.1
6. hCBX8 (E/S) 0.691 1.74 34.5
7. flyPc (E/S) 0.118 1.44 5.9
2. KAH74 (E/X) 0.188 1.37 9.4


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL
10x buffer d.p. 2.0
phophatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations (*Note: use insert + old backbone in calculations)

Ligation Plate results (lig : neg crtl) 11/19/09
1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng KAH86 >10:1 (Pick 2)
2. KAH48(S/P)/ 20 ng  
3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng KAH87 >10:1 (Pick 2)
4. mPcCD(E/S)/3386*, > 42 ng + " KAH88 >10:1 (Pick 2)
5. flyPcCD(E/S)/3386*, > 42 ng + " KAH89 >10:1 (Pick 2)
6. fshPcCD(E/S)/3386*, > 42 ng + " KAH90 >10:1 (Pick 2)
7. hCBX8(E/S)/1170, > 42 ng + " KAH91 >10:1 (Pick 2)
8. hCBX8(E/S)/1170, > 42 ng + " KAH92 >10:1 (Pick 2)
9. KAH74(S/P)/ 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 1.4 --- 1.7 1.7 1.7 1.7 0.5 2.5 ---
Vector DNA 0.6 0.6 2.5 2.5 2.5 2.5 2.5 2.5 ---
2x lgn buf (Roche) 5.0 5.0 5.5 5.5 5.5 5.5 5.5 5.5 5.5
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 2.0 3.4 0.3 0.3 0.3 0.3 1.5 --- 2.0
  10 μL 10 μL 11 μL 11 μL 11 μL 11 μL 11 μL 11.5 μL 11 μL

Oligo annealing

  1. NLS myc stop (KAH011)
  2. K-stop (KAH012)
  3. Adapter BglII/EcoRI
  4. Adapter PstI/BglII


DNA (oligos, 100 μM) up to 18 μL
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight