User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/26

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07/26/11

  • ✓ Cultures/ minipreps: miR-luc 198/MV8 set (3 each, 27 total); DPRE-208/V0200 (4); KAH225 (2); 183-DF4, DF5, DF6, 177/MV8 retransformations (2 each); STOP retransformation (2)



Minipreps
Digests:
1-27. miR-luc 198/MV8 set: ApaLI
28-31. DPRE-208/V0200: SpeI
32, 33. KAH225: E/P
34-41. 183-DF4, DF5, DF6, 177/MV8: ApaLI
42, 43: STOP: E/P

Reagent Volume Expected:
MV8 bands = 1243, 1135, 288
1-3. 186-198/MV8 = 3932*, 2772**, 711
4-6. 184-198/MV8 = 4098*, 2772**, 711
7-9. 187-198/MV8 = 4176*, 2772**, 711
10-12. 188-198/MV8 = 4031*, 2772**, 711
13-15. 189-198/MV8 = 4203*, 2772**, 711
16-18. 190-198/MV8 = 4275*, 2772**, 711
19-21. 191-198/MV8 = 4130*, 2772**, 711
22-24. 183-198/MV8 = 4290*, 2772**, 711
25-27. 192-198/MV8 = 4368*, 2772**, 711
*insert + 234 (vector) + 1673 (198 frag)
**last 198 frag + 1655

28-31. DPRE-208/V0200
DPRE fwd = 6182, 1884
DPRE rev = 4442, 3624
32, 33. KAH225 = 3200, 1494

MV8 bands = 1243, 1135, 288
34, 35. 183-DF4/MV8 = 4218*, 2838**, 711
36, 37. 183-DF5/MV8 = 4218*, 2838**, 711
38, 39. 183-DF6/MV8 = 4218*, 2838**, 711
34, 35. 183-DF4/MV8 = 4218*, 2772**, 711
*insert + 234 (vector) + 1601 (DF frag)
**last DF or 177 frag + 1655

42, 43. STOP = 3200, 13

Miniprep digests 1, 7/26/11
15 μL/lane; 1% agarose

Miniprep digests 2, 7/26/11
15 μL/lane; 1% agarose

Miniprep digests 3, 7/26/11
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.

--> 100% success! Keep one of each successful ligation. Combine retransformation miniprep duplicates (34-43).



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