User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/01

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08/01/11

  • ✓ Assemblies: KAH231, 232, 233; KAH213, 219/MV1; KAH219, 223, 224/pSB31 blunt



Assemblies

  1. KAH231: (1) Kozak/(S/P)/18 + KAH226/(X/P)/1564 ✓
  2. KAH232: " + (2) KAH229/(X/P)/1756
  3. KAH233: " + (3) KAH230/(X/P)/808
  4. KAH213/MV1: (4) KAH213/(E/S)/2834 + MV1/(E/X)/5428 ✓
  5. KAH219/MV1: (5) KAH219/(E/S)/2422 + "
  6. KAH219/pSB31: KAH219/(E/S blunt)/2422 ✓ + pSB31/(E blunt dp)/5127 ✓
  7. KAH223/pSB31: KAH223/(E/S blunt)/1537 ✓ + "
  8. KAH224/pSB31: KAH224/(E/S blunt)/1837 ✓ + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digest 8/01/11
30 μL/lane, 1% agarose
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. Kozak (S/P) 0.461 2.04 23.1
2. KAH229 (X/P) 0.417 1.91 20.9
3. KAH230 (X/P) 0.115 2.53 5.8
4. KAH213 (E/S) 0.132 1.59 6.6
5. KAH219 (E/S) 0.158 2.07 7.9


> Ligations

Ligation Plate results (lig : neg crtl) 08/02/11
1. KAH226(X/P)/1564, 19 ng + Kozak(S/P)/~3218, 20 ng KAH231 >10:1 (Pick 2)
2. KAH229(X/P)/1756, 22 ng + " KAH232 >10:1 (Pick 2)
3. KAH230(X/P)/808, 10 ng + " KAH233 >10:1 (Pick 2)
4. Kozak(S/P)/ 20 ng  
5. KAH213(E/S)/2834, 21 ng + MV1(E/X)/5428, 20 ng KAH213/MV1 5:1 (Pick 3)
6. KAH219(E/S)/2422, 18 ng + " KAH219/MV1 5:1 (Pick 3)
7. MV1(E/X), 20 ng  
8. KAH219(E/S bl)/2422, 19 ng + pSB31(E bl dp)/5127, 20 ng KAH219/pSB31 3:1 (Pick 4)
9. KAH223(E/S bl)/1537, 12 ng + " KAH223/pSB31 3:1 (Pick 4)
10. KAH224(E/S bl)/1837, 14 ng + " KAH224/pSB31 4:1 (Pick 4)
11. pSB31(E bl dp)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10 11
Insert DNA 0.5 1.5 2.0 --- 3.2 2.2 --- 2.0 1.5 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.8 0.8 0.8 0.8
2x lgn buf (Roche) 5.0 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 1.0
dH2O 2.5 1.5 1.0 3.0 0.3 1.3 3.5 1.2 1.7 2.2 3.2
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL



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