Difference between revisions of "Prbbbb:vector pcr"

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*dNTP mix 10mM each nucleotide
*dNTP mix 10mM each nucleotide
*reverse prefix primer [http://brickit.crg.es/registry/biobrick/190/ rg0301 (CRG)] (fusion format, ACCGGTTAATACTAGTAGCGGCC)
*reverse prefix primer [http://brickit.crg.es/registry/biobrick/190/ rg0301 (CRG)] -- [http://partsregistry.org/Part:BBa_J18910 -- BBa_J18910]
*reverse suffix primer [http://brickit.crg.es/registry/biobrick/191/ rg0302 (CRG)] (fusion format, GCCGGCCATCTAGAAGCG)
*reverse suffix primer [http://brickit.crg.es/registry/biobrick/191/ rg0302 (CRG)] (fusion format, GCCGGCCATCTAGAAGCG)
*vector template DNA
*vector template DNA

Revision as of 02:55, 25 January 2010

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A PCR reaction to generate linearized construction vector backbones for Fusion(Freiburg)-formatted Biobrick assemblies.

The classic assembly protocol digests the target vector (usually pSB1*) in parallel to the digestions of the two Biobrick parts. We prefer to create a stock of target backbone by PCR with a fast high-fidelity polymerase. This way, mutations in the ccdB death cassette of the target vector cannot escape selection.


  • 100 ul + PCR tubes
  • Phusion HotStart Polymerase 2 U/ul
  • Phusion HF Buffer 5x
  • dNTP mix 10mM each nucleotide
  • ddH2O
  • reverse prefix primer rg0301 (CRG) -- -- BBa_J18910



setup PCR reaction

µl, single reaction 3.5xMaster 10xMaster
H2O 76µl 266 760
5x HF Buffer 20µl 70 200
10mM dNTP 2µl 7 20
rg0301 100 µM 0.5µl 1.75 5
rg0302 100 µM 0.5µl 1.75 5
Phusion 1µl 3.5 10
template DNA 0.1µl -- --

PCR program

  • 30"@98C;
  • 35x (10"@98°C; 15"@68C; 1'30"@72C);
  • 10'@72C;
  • inf. 4C


  1. add 1µl DpnI, incubate for 1h @ 37°C
  2. heat-inactivate 20'@80°C
  3. purify with PCR purification kit
    • elute in water **not** elution buffer
  4. dilute to 28 ng/µl (we assume a 10 % dilution by the following Phosphatase treatment)
  5. proceed with restriction C
    • add 2µl restriction mix C to each 8µl DNA
    • incubate for 3h @ 37°C
    • heat-inactivate 20' @ 80°C
  6. proceed with phosphatase treatment
    • add 10 x antarctic phospatase buffer to 1 x final concentration
    • add 1µl Antarctic Phosphatase
    • incubate 1h @ 37°C; heat-inactivate 5' @ 65°C
  7. verify samples on 1% Agarose gel


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