User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/06

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Karmella's BioBrick Cloning Main project page
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05/06/15

  • Ryan - minipreps & digests for promoter/Regulator/MRV clones



Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • Cut w/ E/KpnI
    • See my version of MRV map: https://benchling.com/s/oJ73uatK/edit
    • Note: BbsI drop-in of promoter knocks out KpnI site (54); no E, X, or S sites are restored by promoter insert
    • Note: EcoRI (1025) and XbaI (1040) were maintained after Regulator insertion
  1. pAub-AubR/MRV
  2. pBja-BjaR/MRV
  3. pBra-BraR/MRV
  4. pRpa-RpaR/MRV


Reagent Volume Expected:
1,2. pAub/AubR/MRV = 4240
3,4. pBja/BjaR/MRV = 4099
5,6. pBra/BraR/MRV = 4191
7,8. pRpa/RpaR/MRV = 4116
9. Empty MRV = 2230, 971

np pro. Reg/MRV = ~4000, 971
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
KpnI 1.0
dH2O 8.5
  15 μL

--> 37°C/ ~10 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pAub-AubR/MRV-1 0.152 1.930 152.3
2. pAub-AubR/MRV-2 0.079 1.992 78.6
3. pBja-BjaR/MRV-1 0.093 1.989 92.7
4. pBja-BjaR/MRV-2 0.043 1.934 43.1
5. pBra-BraR/MRV-1 0.070 2.021 69.9
6. pBra-BraR/MRV-2 0.030 1.946 29.8
7. pRpa-RpaR/MRV-1 0.093 1.956 92.6
8. pRpa-RpaR/MRV-2 0.033 2.000 32.7


Conclusions

  • Yields low b/c cultures were only grown for ~7 hours
  • Lane 1: looks like AubR/MRV (modular receiver vector) without a promoter insert
  • Lanes 2 - 8: very confusing, cannot interpret this with current information
  • Lane 9: appears as expected for empty MRV
  • Re-do dig/lig, include negative controls for each unique vector



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