MEGAWHOP

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

Designing Primers

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

-- SteveSowa - 25 Apr 2012