User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/07

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05/07/15

  • Ryan - Dig/Lig reaction do-over



Ryan - Dig/Lig reaction do-over

  • This time include neg ctrls for each reaction
  • Dig/Lig reactions
    • 2:1 ratio calculation: ~150 bp insert / ~4000 bp vector * 2 * 100 = 3.75 ng insert
  1. pAub-PCR(E/S dp) + AubR/MRV
  2. AubR/MRV
  3. pBja-PCR(E/S dp) + BjaR/MRV
  4. BjaR/MRV
  5. pBra-PCR(E/S dp) + BraR/MRV
  6. BraR/MRV
  7. pRpa-PCR(E/S dp) + RpaR/MRV
  8. RpaR/MRV


Reagent Rxn Mix (x9)
100 ng Vector ### (up to 13.5) ---
insert 1.0 ---
10x FD buf 2.0 18.0
10 mM DTT 1.0 9.0
10 mM ATP 1.0 9.0
FastDigest BbsI/BpiI 1.0 9.0
Roche T4 DNA ligase 0.5 4.5
dH2O ### ---
  20.0

--> Pipette 5.5 μL master mix into each PCR tube
--> Add 1.0 μL insert into each tube
--> Add 100 ng vector DNA
--> Add x μL dH2O = 13.5 - vector volume
--> Mix by flicking

LabNet OptiMax Thermocycler: BbsI Dig/Lig

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


Transformation(s)

  • Thaw 2 tubes chem competent DH5α-turbo on ice
  • Transfer 10.0 μL each ligation (1/2 rxn.) to fresh sterile 0.5 mL tube
  • Add 50 μL DH5α-turbo; pipette up-and-down 3x GENTLY
  • Incubate 5 min. on ice
  • Plate on 100 μg/mL amp


RESULTS (5/08/15)

  • ~25 colonies on each ligation plate, and same number on negative controls
  • Rene has observed something similar for dig/lig.
  • Pick grow up liquid cultures for two colonies from each plate on Monday




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