Difference: MEGAWHOP (3 vs. 4)

Revision 42012-05-01 - BrianRenda

 
META TOPICPARENT name="ProtocolList"

Megaprimer whole plasmid cloning

aka MEGAWHOP cloning
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

genome_mod_figure.JPG

Designing Primers

primers.png

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
Changed:
<
<		Set elongation time according to size of insert
>
>		Set elongation time according to size of insert.
 
Changed:
<
<		Purify PCR products
>
>		Purify PCR products. This can either be done through a gel extraction or by adding Dpn1 directly to the Phusion reaction mixture after PCR, digesting at 37°C for 1 hour, and then doing a standard PCR clean up. Dpn1 works efficiently in a Phusion reaction mixture.
 

PCR Recombinant Plasmid

Use modified 10ul Phusion (or other high fidelity polymerase) protocol
  • 2 ul 5x Buffer
  • 1 ul dntps
Changed:
<
<
  • 500 ng PCR insert product
>
>
  • 500 ng PCR insert product (Note, for optimal results, have a 250-fold molar excess of your megaprimer to your target plasmid).
 
  • ~10 ng target plasmid
  • ddH20 to 9.5 ul
  • then add 0.5 ul Phusion
Changed:
<
<		Adjust Elongation time for the length of the entire plasmid
>
>		Adjust Elongation time for the length of the entire plasmid (90 seconds per kb).
 

Digest

Changed:
<
<		Digest Recombinant Plasmid PCR product with 0.5 ul Dpn1 at 37°C for one and a half hours to remove parental DNA
>
>		Once the reaction is complete, digest the Recombinant Plasmid PCR product with 0.5 ul Dpn1 at 37°C for one and a half hours to remove parental DNA.
 

Transform

Changed:
<
<		Electroporate the new plasmid into E coli
>
>		Heatshock 5ul of the Dpn1-digested MEGAWHOP reaction mixture into 25ul of chemically competent E coli.
 

-- SteveSowa - 25 Apr 2012

Deleted:
<
<
 
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