User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/10

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04/10/10

  • ✓ Assemblies: re-digest vectors and KAH96 from yesterday; re-do ligations



Assemblies
--> See 4/09/10

> Digests (Fermentas FD)
--> Repeat digests from yesterday; increase time to 1 hour:

  1. KAH96 (E/S/ApaLI)
  2. KAH145 (E/X)
  3. KAH116 (E/X)
Reagent 2,3 1 Cloning re-digest 4/10/10
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL up to 24 μL
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ApaLI --- 1.0
dH2O --- ---
  30 μL 30 μL --> 37°C/ ~ 1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH96 (E/S) 0.987 1.88 49.3
2. KAH145 (E/X) 0.507 1.72 25.3
3. KAH116 (E/X) 0.539 1.90 27.0


> Ligations

Ligation Plate results (lig : neg crtl) 04/10/10
1. KAH96(E/S)/2825, 33 ng + KAH145(E/X)/6755, 40 ng KAH146 10:1 (Pick 2)
2. KAH142(E/S)/4571, 54 ng + " KAH147 10:1 (Pick 2)
3. KAH145(E/X)/ 40 ng ~5 colonies
4. KAH01(E/S)/186, 2 ng + KAH116(E/X)/3975, 20 ng KAH148 10:1 (Pick 2)
5. KAH02(E/S)/186, 2 ng + " KAH149 10:1 (Pick 2)
6. KAH03(E/S)/186, 2 ng + " KAH150 10:1 (Pick 2)
7. KAH04(E/S)/186, 2 ng + " KAH151 10:1 (Pick 2)
8. KAH06(E/S)/1170, 12 ng + " KAH152 10:1 (Pick 2)
9. KAH06(E/S)/1173, 12 ng + " KAH153 10:1 (Pick 2)
10. KAH116(E/X)/ 50 ng ~10 colonies
  1 2 3 4 5 6 7 8 9 10
Insert DNA 0.7 2.7 --- 0.5 0.5 0.5 0.5 0.5 1.5 ---
Vector DNA 1.6 1.6 1.6 0.7 0.7 0.7 0.7 0.7 0.7 0.7
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 1.7 --- 2.4 2.8 2.8 2.8 2.8 2.8 1.8 3.3
  10 μL ~10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


--> R.T./ ~10 min.; Add to 20 μL Z-DH5α

--> 4/11/10: Success! Pick 2 colonies each.


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