Prbbbb:vector pcr

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Contents

Overview

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.

Materials

Procedure

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

Post-Processing

  1. add 1µl DpnI, incubate for 1h @ 37°C
    • mix the tube so that the enzyme contacts all surfaces
    • longer (2h) incubation may still reduce background
  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

Notes

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