User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/28

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04/28/15

  • Ryan - minipreps & digests
  • Ryan - PCR & Dig/Lig (promoter insert trial #2)



Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • BraR/MRV colonies - picked 4 additional colonies from original cloning plate
  • Cut w/ E/X
  1. Bra/MRV 3
  2. Bra/MRV 4
  3. Bra/MRV 5
  4. Bra/MRV 6
  • promoter/Regulator/MRV colonies
  • Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
  1. pAub/AubR/MRV
  2. pBja/BjaR/MRV
  3. pRpa/RpaR/MRV


Reagent Volume Expected:
1-4. Bra/MRV = ~3200, 776
Empty MRV E/X = ~3200
5. pAub/AubR/MRV = 4240
6. pBja/BjaR/MRV = 4099
7. pRpa/RpaR/MRV = 4116
Empty vector = ~4000, 971
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
BraR/MRV #5 0.465 1.913 465.4


Conclusions

  • BraR/MRV - Success! Keep colony 5 (lane 3)
  • promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts



Ryan - PCR & Dig/Lig (promoter insert trial #2)

Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)

  • 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
    • PCR-amplify promoter inserts (same primers as Stage 1)
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Assemblies

  1. pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
  2. pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
  3. pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
  4. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980


  • Measure conc.'s
    • Values from last week, except BraR/MRV, which was measured today
Sample OD260 260/280 ng/μL
1. AubR/MRV 0.258 1.906 257.7
2. BjaR/MRV 0.177 1.904 177.3
3. BraR/MRV #5 0.465 1.913 465.4
4. RpaR/MRV 0.246 1.896 246.2
  • PCR-amplify promoters
    • 2x 50 μL rxns each
  1. pAubR, EcoRI fwd, XbaI rev
  2. pBjaR, EcoRI fwd, XbaI rev
  3. pBraR, EcoRI fwd, XbaI rev
  4. pRpaR, EcoRI fwd, XbaI rev
Reagent Vol Mix (x2) Expected:
1. pAubR = 153
2. pBjaR = 122
3. pBraR = 214
4. pRpaR = 136
Hover name
5 μL/lane; 1% agarose; Ladder
gBlock DNA (2 ng/μL) 0.5 1.0
10 μM XbaI rev 1.0 2.0
10 μM EcoRI fwd 1.0 2.0
2x GoTaq green 25.0 50.0
dH2O 22.5 8.7
  50.0 μL

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞