User:Karmella Haynes/Notebook/BioBrick cloning/2015/10/20

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Karmella's BioBrick Cloning Main project page
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10/20/15

  • Cas-tone: Digest checks on H3 constructs
  • Cas-tone: sequencing verification



Cas-tone: Digest checks on H3 constructs

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Same as 06/24/15
    • Check with SpeI/HindIII digests
    • Note: X/N H3 insert destroys SpeI site. Fragment without H3 part (Hind-Hind large) = 7960 (~8.0); add H3-1-# size + 8.0 to get length for longer H/H band
    • H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433
Reagent Volume Expected:
1,2. H3-1-40_MV12 = 2.3, 8.1
3,4. H3-1-60_MV12 = 2.3, 8.2
5,6. H3-1-80_MV12 = 2.3, 8.3
7,8. H3-1-116_MV12 = 2.3, 8.4
9,10. H3-1-136_MV12 = 2.3, 8.4
Empty MV12 = 6.9, 2.3, 1.0
Hover name
15 μL/lane; 1% agarose; Ladder = 3 μL
DNA(plasmid) 2.0 μL
10X buffer 1.5
SpeI 1.0
HindIII 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.


  • DNA concentrations: BioTek Synergy H1 Take 3
Sample OD260 260/280 ng/µL
1. H340_MV12 0.071.81270.263
2. H340_MV12 b0.061.91559.669
3. H360_MV12 a0.0721.88271.57
4. H360_MV12 b0.0521.92551.593
5. H380_MV12 a0.0721.81872.02
6. H380_MV12 b0.0291.81428.636
7. H3116_MV12 a0.0371.86536.735
8. H3116_MV12 b0.0561.84456.147
9. H3136_MV12 a0.0711.8470.707
10. H3136_MV12 b0.0411.92640.657


CONCLUSION

  • Success! Clones 1-10 look correct.
  • Verify via sequencing
  • Sequencing order (10/21/15) - #
  1. H340-MV12-a, CMVf
  2. H340-MV12-a, DD123
  3. H340-MV12-b, CMVf
  4. H340-MV12-b, DD123
  5. H360-MV12-a, CMVf
  6. H360-MV12-a, DD123
  7. H360-MV12-b, CMVf
  8. H360-MV12-b, DD123
  9. H380-MV12-a, CMVf
  10. H380-MV12-a, DD123
  11. H380-MV12-b, CMVf
  12. H380-MV12-b, DD123
  13. H3116-MV12-a, CMVf
  14. H3116-MV12-a, DD123
  15. H3116-MV12-b, CMVf
  16. H3116-MV12-b, DD123
  17. H3136-MV12-a, CMVf
  18. H3136-MV12-a, DD123
  19. H3136-MV12-b, CMVf
  20. H3136-MV12-b, DD123



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