User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23

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Karmella's BioBrick Cloning Main project page
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04/23/15

  • Ryan - Receiver cloning stage 2
  • Rene - sequencing for g053-2, g054



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


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
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. RpaR/MRV 0.246 1.896 246.2


  • Digests (Fermentas FD)
    • E/X
    • Inserts are gBlocks = 2.0 ng/μL
Reagent Volume
DNA (20 ng) 10.0 μL
10x buffer 2.0
EcoRI 1.0
XbaI 1.0
dH2O 6.0
  20.0 μL --> 37°C/ ~15 min.


  • Dephosphorylation (Roche)
    • Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions Link
Reagent Volume
DNA (digest) 20.0 μL (20 ng)
phosphatase 1.0
dH2O ---
  20 μL

--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL


  • Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure)
    • Inserts are gBlocks = ~2ng/μL
    • For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
  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. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
  • Note: BraR cloning was not successful, omit from this round of assembly
  1 2 3
Insert DNA 5.0 5.0 5.0
Vector DNA 0.2 0.3 0.2
10x FD buf 2.0 2.0 2.0
10 mM DTT 1.0 1.0 1.0
10 mM ATP 1.0 1.0 1.0
FD BbsI/BpiI 1.0 1.0 1.0
T4 ligase (Roche) 1.0 1.0 1.0
dH2O 8.8 8.7 8.8
  20.0 μL 20.0 μL 20.0 μL

Thermal cycler: Labnet - BbsI Dig/Lig

  • 6x[37°C, 5 min.; 23°C 5 min.]
  • 4°C, ∞



Rene - sequencing

  1. g53-2, f (P139) - Confirmed
  2. g53-2, r (P140) - low Phred 20 score
  3. g54, f (P139) - Confirmed
  4. g54, r (P140) - low Phred 20 score




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