Latest Posts

All Posts

Tips for Titering Your Lentiviral Preps

Posted by Meghan Rego on Mar 15, 2016 10:30:00 AM

The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time to move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to move a project forward, transductions should not be rushed into. Before you start any transduction, you should always titer your virus - that is determine the amount of virus you actually have in your prep. Taking time to properly titer your virus will not only ensure that your infection is designed in the best possible way but it may also save you time in the long run. Read on for an overview of the titering options and the benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here refer to
Kutner et. al.).

Read More >

Topics: Techniques, Viral Vectors

PITChing MMEJ as an Alternative Route for Gene Editing

Posted by Mary Gearing on Feb 23, 2016 10:30:00 AM

If you follow CRISPR research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small homology regions (5-25 bp) for repair, making it easier to construct targeting vectors. Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for complicated cloning of homology arms.

Read More >

Topics: Plasmid Technology, Genome Engineering, CRISPR, Techniques

REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

Posted by Mary Gearing on Feb 10, 2016 10:30:00 AM

Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular biology and genetics. It can be used to characterize the function of certain regions in a promoter or gene, as well as to study the effects of inactivating/activating mutations. In biomedical research, modeling patient mutations using SDM can help determine if a variant is causal for a given disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While commercial kits are available for making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency as Gibson Assembly and GeneArt cloning - but at a much lower cost. Read on to find out how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized Plasmids After PCR).

Read More >

Topics: Protocols, Techniques, Plasmid Cloning

Illuminating Epigenetics with A FRET Based Biosensor

Posted by Emma Markham on Nov 19, 2015 10:30:00 AM

Epigenetics has recently been hitting the headlines, with sotires like the potential devastation of the palm oil industry through epigenetic effects on the Cover of Nature. So what is epigenetics and what tools are available to study it?

Read More >

Topics: Hot Plasmids, Fluorescent Proteins, Techniques

The Materials Science of Optogenetics Experiments

Posted by Guest Blogger on Sep 17, 2015 10:30:00 AM

This post is part of our Primer on Optogenetics and was contributed by guest blogger Derek Simon.

The surgeries and standard molecular neuroscience validation experiments we discussed last week are only half of the battle when using optogentics to answer a research question. The flip side of the optogenetics coin is materials science-based. Light is delivered to your opsin through a small piece of fiber optic cable implanted into the animal’s skull (right). The fiber optic cable is threaded throughand fixed to—an optical insulator called a ferrule (below). The fiber optic cable/ferrule is inserted into the target brain region using stereotaxic surgery and cemented to the animal’s skull using dental cement (a similar procedure as implanting a guide cannula). A fiber optic patch cable is then connected from laser to ferrule to deliver light pulses to the target brain region.

Read More >

Topics: Optogenetics, Techniques, Primer on Optogenetics

Blog Logo Vertical-01.png

Subscribe to Our Blog