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Site Directed Mutagenesis by PCR

Posted by Guest Blogger on Aug 2, 2016 10:30:00 AM

This post was contributed by guest blogger, Kristian Laursen from Cornell University.

Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage.

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Topics: Plasmid Protocols and Tips, PCR, Plasmids

Plasmids 101: Colony PCR

Posted by Beth Kenkel on May 12, 2016 10:30:00 AM

This post was contributed by guest blogger Beth Kenkel, a Research Assistant in the Department of Pediatrics at the University of Iowa. If you're interested in guest blogging, let us know!

Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however colony PCR can accomplish the same thing in less time and for less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze product size. This blog post discusses some of the key things to consider when performing colony PCR.

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Topics: Plasmids 101, Plasmid Protocols and Tips, PCR, Plasmids

Plasmid Cloning by PCR

Posted by Various Addgenies on Mar 29, 2016 10:30:00 AM

In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest. You can use similar processes to add overhangs to your insert of interest for Gibson assembly. The steps following primer design and the PCR process itself are very similar to those outlined in our restriction cloning post with a few quirks specific to the PCR cloning process - please check out that post if you need a more detailed refresher on the downstream steps.

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Topics: Plasmid Cloning, PCR, Plasmids

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