---+ Megaprimer whole plasmid cloning aka <nop>MEGAWHOP cloning %BR% aka Overlap Extension PCR cloning Steve Sowa 4/25/2012 Adapted from Bryksin AV, Matsumura I. 2010. Overlap extension PCR Cloning: a simple and reliable way to create recombinant plasmids. _Biotechniques_.48(6):463-5. ---++ Purpose To insert a DNA sequence into a plasmid without restriction enzymes. ---++ Experimental Steps * Create primers that amplify region of interest and hybridize with target plasmid * Perform a Phusion PCR with primers using the region of interest as template * Perform a second Phusion PCR using the products of the first PCR and the target plasmid as template * Digest Second PCR with Dpn1 to remove parental plasmid * Transform in _E coli_ <img src="%ATTACHURLPATH%/model.png" alt="model.png" width='549' height='103' /> ---++ Designing Primers <img src="%ATTACHURLPATH%/primers.png" alt="primers.png" width='374' height='204' /> Primers need to have two components * a region that amplifies the insert (A(or B), 20-25nt) * a region that targets the new plasmid (C(or D), 30-40nt) The target plasmid regions should preferably be 50-200nt apart (C to D). Order (A+C) primer and (B+D) primer. ---++ PCR Insert Use stardard 25ul Phusion (or other high fidelity polymerase) protocol * PCR 1(25ul reaction) * 5 ul 5x Buffer * 1.5 ul dntps * 1.25 ul primer (A+C) * 1.25 ul primer (B+D) * x ul template plasmid (<250ng) * ddH20 to 24.5 ul * then add 0.5 ul Phusion Set elongation time according to size of insert Purify PCR products ---++ PCR Recombinant Plasmid Use modified 10ul Phusion (or other high fidelity polymerase) protocol * 2 ul 5x Buffer * 1 ul dntps * 500 ng PCR insert product * ~10 ng target plasmid * ddH20 to 9.5 ul * then add 0.5 ul Phusion Adjust Elongation time for the length of the entire plasmid ---++ Digest Digest Recombinant Plasmid PCR product with 0.5 ul Dpn1 at 37°C for one and a half hours to remove parental DNA ---++ Transform Electroporate the new plasmid into _E coli_ -- Main.SteveSowa - 25 Apr 2012
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Topic revision: r2 - 2012-04-26 - JeffreyBarrick