User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/12

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05/12/15

  • Cas-tone - streak Addgene plasmids for single colonies; design histone primers
  • Ryan - re-treat insert DNA (heat, slow anneal); redo dig/lig
  • Minipreps - KAH87/MV10 from 2x 5 mL cultures (CMV-PcTF for Ben)



Ryan - retry dig/lig

  • Idea: small inserts may have melted and re-annealed improperly during 80°C enzyme deactivation
  • Use remaining PCR reaction for another digest/phosphatase treatment.
  • Do all steps in thermal cycler


  • Digest & dephos PCR fragments
Reagent Volume
DNA (500 ng) 10.0 μL
10X buffer 2.0
EcoRI 1.0
XbaI 1.0
SAP (Roche) 1.0
dH2O 5.0
  20.0


  • LabNet OptiMax Thermocycler: AnOlig RD
    • 37°C, 10 min
    • 95°C, 5 min
    • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
    • 25°C, ∞


  • 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. pBja-PCR(E/S dp) + BjaR/MRV
  3. pBra-PCR(E/S dp) + BraR/MRV
  4. pRpa-PCR(E/S dp) + RpaR/MRV


Reagent Rxn Mix (x5)
100 ng Vector ### (up to 12.5) ---
insert 2.0 ---
10x FD buf 2.0 10.0
10 mM DTT 1.0 5.0
10 mM ATP 1.0 5.0
FastDigest BbsI/BpiI 1.0 5.0
Roche T4 DNA ligase 0.5 2.5
dH2O ### ---
  20.0

--> Pipette 5.5 μL master mix into each PCR tube
--> Add 2.0 μL insert into each tube
--> Add 100 ng vector DNA
--> Add x μL dH2O = 12.5 - 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 (5/13/15)

  • ~15 colonies per plate
  • Pick two colonies from each for 5 mL cultures