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Plasmids 101: Control Plasmids

Posted by Chari Cortez on Apr 9, 2015 11:29:00 AM

There are many, many different types of experiments carried out by scientists every day. Although the designs and outcomes may vary, one thing should be present in every experiment-based investigation of a hypothesis: proper controls!

For every experiment, an investigator needs a standard against which the results can be compared; results from an experiment lacking the proper controls are invariably inconclusive and unreliable. Proper controls provide the constant variables that enable the correct interpretation of the effect of the independent variable you are testing. Importantly, they demonstrate the functionality of your experimental system and help identify opportunities for troubleshooting or optimization within your experiment. Read on to learn more about the various controls that can be used for plasmid-based experiments.

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Topics: Lab Tips, Plasmids 101

CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

Posted by Guest Blogger on Feb 18, 2015 10:09:22 AM

The following post was contributed by Daniel Bauer and Matthew Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint of the CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations behind this research.

 

Using CRISPR/Cas9 for Targeted Genomic Deletions

We were inspired to produce intrachromosomal deletions based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One observation was an inverse relationship between deletion size and frequency [3].

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Topics: Genome Engineering, Lab Tips, CRISPR, Protocols

Plasmids 101: E. coli Strains for Protein Expression

Posted by Julian Taylor-Parker on Feb 10, 2015 10:06:00 AM

In a previous Plasmids 101 blog, we reviewed the salient features of several popular strains of E. coli for DNA propagation. While great for cloning purposes, these E. coli strains are not usually well suited for recombinant protein expression. Many challenges can arise when over-expressing a foreign protein in E. coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular commercial strains designed to avoid them.

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Topics: Lab Tips, Plasmids 101

22 Hot Plasmid Technologies from 2014

Posted by Joanne Kamens on Jan 6, 2015 12:21:53 PM

Updated Mini-transposon Vector for Bacterial Mutagenesis or Gene Targeting

Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other markers into a target genome. They did this by selecting the important elements from existing transposon and vector systems and creating an all-synthetic vector that included only the elements needed for function.

The lab validated this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the genome. Although this tool was published in 2011, it was only recently made available through Addgene and we want to highlight it for use in your research.

Check out Joanne's Reddit AMA

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Topics: Hot Plasmids, Lab Tips, Plasmid Kits

Plasmids 101: Common Lab E. coli Strains

Posted by Matthew Ferenc on Nov 7, 2014 9:56:00 AM

This post was updated on Nov 14, 2017.

You've worked hard designing your plasmid – you carefully selected the optimal promoter for your gene of interest, painstakingly cloned into the perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent protein downstream, separated by an IRES element. You did a lot of work! But let’s take a moment to recognize your little prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria.

It’s hard to count the number of different commercial strains of E. coli currently available  a quick Google search suggests there are hundreds. This only includes general lab strains designed for subcloning or protein expression. If you were to include customized strains, the number is probably in the thousands! The goal of this article is to provide enough background for you to distinguish the features of any common lab strain and determine whether it is appropriate for propogating your plasmid or carrying out your experiment.

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Topics: Lab Tips, Plasmids 101

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