User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/19

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07/19/11

  • ✓ Cultures/minipreps: started cultures last night, KAH219, 221, 222, 223, 224 (4 each)
  • ✓ Assemblies: KAH213, 214, 215, 219, 220, 221/MV1
  • ✓ Cultures: KAH219, 223, 224/pSB31 (8 each) (o/n)
  • ✓ Cultures: KAH220, 221, 222/pSB31 (5 ml, 2 each) (o/n)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
pSB31 = 4500, 450

*pSB31 bands
**last frag + 300
1-4. 219/pSB31 5-8 = 4500*, 150*, 444, 1735, 532**
5-8. 221/pSB31 5-8 = 4500*, 150*, 444, 1490**
9-12. 222/pSB31 5-8 = 4500*, 150*, 636, 1735, 532**
13-16. 223/pSB31 5-8 = 4500*, 150*, 636, 1190**
17-20. 224/pSB31 5-8 = 4500*, 150*, 636, 1490**
File:KAH 071911 gel1.tif
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.

--> 221/pSB31 and 222/pSB31 worked. Pick more colonies for others.


Assemblies

  1. KAH213/MV1: (1) KAH213/(E/S)/2834 + MV1/(E/X)/5428
  2. KAH214/MV1: (2) KAH214/(E/S)/1757 + "
  3. KAH215/MV1: (3) KAH215/(E/S)/2057 + "
  4. KAH219/MV1: KAH219/(E/S)/2422 + "
  5. KAH220/MV1: KAH220/(E/S)/1345 + "
  6. KAH221/MV1: KAH221/(E/S)/1757 + "
  7. KAH222/MV1: KAH222/(E/S)/2614 + "
  8. KAH223/MV1: KAH223/(E/S)/1537 + "
  9. KAH224/MV1: KAH224/(E/S)/1837 + "


> Digests (Fermentas FD)

Reagent Volume   File:KAH 071911 gel2.tif
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. KAH213 (E/S) 0.174 1.88 8.7
2. KAH214 (E/S) 0.227 1.84 11.3
3. KAH215 (E/S) 0.206 2.61 10.3


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH213(E/S)/2834, 26 ng + MV1(E/X)/5428, 25 ng KAH213/MV1 #:1 (Pick #)
2. KAH214(E/S)/1757, 16 ng + " KAH214/MV1 #:1 (Pick #)
3. KAH215(E/S)/2057, 19 ng + " KAH215/MV1 #:1 (Pick #)
4. KAH219(E/S)/2422, 22 ng + " KAH219/MV1 #:1 (Pick #)
5. KAH220(E/S)/1345, 12 ng + " KAH220/MV1 #:1 (Pick #)
6. KAH221(E/S)/1645, 15 ng + " KAH221/MV1 #:1 (Pick #)
7. KAH222(E/S)/2614, 24 ng + " KAH222/MV1 #:1 (Pick #)
8. KAH223(E/S)/1537, 14 ng + " KAH223/MV1 #:1 (Pick #)
9. KAH224(E/S)/1837, 17 ng + " KAH224/MV1 #:1 (Pick #)
10. MV1(E/X)/ 25 ng  
  1 2 3 4 5 6 7 8 9 10
Insert DNA 3.0 1.4 1.8 1.6 0.5 0.6 1.1 1.1 0.7 ---
Vector DNA 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6
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 0.6 2.2 1.8 2.0 3.1 3.0 2.5 2.5 2.9 3.6
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


--> Add 30 DH5α Turbo; plate on 100 μg/mL amp