Prbbbb:vector pcr

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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

 * 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 (RFC 25 format, ACCGGTTAATACTAGTAGCGGCC)
 * reverse suffix primer rg0302 (CRG) -- BBa_J18911(RFC 25 format, GCCGGCCATCTAGAAGCG)
 * vector template DNA
 * pSB1AC3F (CRG) -- BBa_J18901
 * pSB1AK3F (CRG) -- BBa_J18902
 * pSB1AT3F (CRG) -- BBa_J18903
 * DpnI restriction enzyme
 * Antarctic Phosphatase and 10 x buffer

Procedure
setup PCR reaction

PCR program


 * 30"@98C;
 * 35x (10"@98&deg;C; 15"@68C; 1'30"@72C);
 * 10'@72C;
 * inf. 4C

Post-Processing


 * 1) add 1µl DpnI, incubate for 1h @ 37&deg;C
 * 2) * mix the tube so that the enzyme contacts all surfaces
 * 3) * longer (2h) incubation may still reduce background
 * 4) heat-inactivate 20'@80&deg;C
 * 5) purify with PCR purification kit
 * 6) *elute in water **not** elution buffer
 * 7) dilute to 28 ng/µl (we assume a 10 % dilution by the following Phosphatase treatment)
 * 8) proceed with restriction C
 * 9) * add 2µl restriction mix C to each 8µl DNA
 * 10) * incubate for 3h @ 37°C
 * 11) * heat-inactivate 20' @ 80°C
 * 12) proceed with  phosphatase treatment
 * 13) * add 10 x antarctic phospatase buffer to 1 x final concentration
 * 14) * add 1µl Antarctic Phosphatase
 * 15) * incubate 1h @ 37°C; heat-inactivate 5' @ 65°C
 * 16) verify samples on 1% Agarose gel

Contact

 * Raik

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