User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/07

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06/07/11

  • ✓ Cultures/minipreps/digests: miR sensor test constructs (1 each of ###/196 set, 2 each of ###/177 set, 1 each of ###/198 set)



Minipreps
> Check with E/ApaLI digests
> mCherry has ApaLI site* (will cut within RFP-Repressor genes, see 6/04/11)

Reagent Volume
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.
Expected:
MV8 bands = 1243, 1135, 288, 234...

196's = CFP+1437, 711*, 2772*
1. 186 = 3462
2. 184 = 3628
3. 187 = 3706
4. 188 = 3561
5. 189 = 3733
6. 190 = 3805
7. 191 = 3660
8. 183 = 3820
9. 192 = 3898

177's = CFP+1609, 711*, 2772*
10,11. 186 = 3634
12,13. 187 = 3878
14,15. 188 = 3733
16,17. 189 = 3905
18,19. 190 = 3977
20,21. 191 = 3832
22,23. 192 = 4070

198's = CFP+1681, 711*, 2772*
24. 186 = 3706
25. 184 = 3872
26. 187 = 3950
27. 188 = 3805
28. 189 = 3977
29. 190 = 4049
30. 191 = 3904
31. 183 = 4064
32. 192 = 4142
  File:KAH 060711 gel1.tif
15 μL/lane; 1% agarose
File:KAH 060811 gel1.tif
Two other #9 colonies, 6/8/11


--> Pick and prep 2 new 192/196's (#9) (tomorrow)
--> For all others, inserts appear to be correct, but there should be a total of 7 bands for all constructs. Confirm via sequencing (tomorrow).