Difference: MEGAWHOP (9 vs. 10)
Revision 102017-06-13 - GabrielSuarez
Megaprimer whole plasmid cloning | |||||||||
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| < < | aka MEGAWHOP cloning | ||||||||
| > > | aka MEGAWHOP cloning aka Overlap Extension PCR cloning | ||||||||
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| < < | aka Overlap Extension PCR cloning | ||||||||
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.
PurposeTo insert a DNA sequence into a plasmid without restriction enzymes.Experimental Steps
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Designing Primers | |||||||||
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Primers need to have two components
PCR Insert | |||||||||
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| < < | Use stardard 25ul Phusion (or other high fidelity polymerase) protocol | ||||||||
| > > | Use standard 25ul Phusion (or other high fidelity polymerase) protocol | ||||||||
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| < < | 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. | ||||||||
| > > | 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 | |||||||||
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| < < | Use modified 10ul Phusion (or other high fidelity polymerase) protocol | ||||||||
| > > | Use modified 10ul Phusion (or other high fidelity polymerase) protocol | ||||||||
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| < < | Adjust Elongation time for the length of the entire plasmid (90 seconds per kb). | ||||||||
| > > | Adjust Elongation time for the length of the entire plasmid (90 seconds per kb). | ||||||||
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| > > | Use modified 25ul Phusion protocol | ||||||||
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| < < | Use modified 25ul Phusion protocol | ||||||||
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| < < | 68°C 5min+98°C 3min+(98°C 30s+68°C 30s+72°C X min)*30 +72°C 10min | ||||||||
| > > | 68°C 5min+98°C 3min+(98°C 30s+68°C 30s+72°C X min)*30 +72°C 10min Adjust Elongation time for the length of the entire plasmid (30 seconds per kb). | ||||||||
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| < < | Adjust Elongation time for the length of the entire plasmid (30 seconds per kb). | ||||||||
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| < < | DigestOnce 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. DpnI Digest | ||||||||
| > > | DigestOnce 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. DpnI Digest | ||||||||
TransformHeatshock 5ul of the Dpn1-digested MEGAWHOP reaction mixture into 25ul of chemically competent E coli. | |||||||||
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ExampleChange the promoter for sgRNA using MegaWHOP. Template sequence: https://benchling.com/s/g4S95i24 | |||||||||
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| UPCASE LETTERS: a region that amplifies the insert (A(or B) | |||||||||
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| < < | lower case letters: a region that targets the new plasmid (C(or D) | ||||||||
| > > | lower case letters: a region that targets the new plasmid (C(or D) | ||||||||
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| < < | *PCR MEGA_primer: | ||||||||
| > > | *PCR MEGA_primer:  | ||||||||
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| < < | *PCR Recombinant Plasmid: | ||||||||
| > > | *PCR Recombinant Plasmid:  | ||||||||
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