User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17

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Karmella's BioBrick Cloning Main project page
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04/17/15

  • Rene - gRNA plasmids, 5 mL cultures (11 total)
  • Ryan - Receiver plasmids, assembly
  • LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer



Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Phusion PCR

  1. AubR, EcoRI F/ XbaI R
  2. BjaR, "
  3. BraR, "
  4. RpaR, "
Reagent Volume Mix (x5) Expected:
1. AubR = 851
2. BjaR = 776
3. BraR = 776
4. RpaR = 779
Hover name
5 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL ---
10 μM F primer 1.0 5.0
10 μM R primer 1.0 5.0
10 mM dNTPs 1.0 5.0
Phusion Pol. 0.5 2.5
5X HF buffer 10.0 50.0
dH2O 36.0 180.0
  50.0


Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • All four worked. Proceed to next step



LCR Development

  • Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer


Phusion PCR

Reagent Rxn1,2 Expected:
1,2. MV10 = 5191
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(clean linear) 1.0 μL
10 μM F primer 1.0
10 μM R primer 1.0
10 mM dNTPs 1.0
Phusion Pol. 0.5
5X HF buffer 10.0
dH2O 35.5
  50.0


Program: Phusion (block A)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Big improvement. Keep this for potential cloning
  • Also try again with higher annealing temp



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