...year and happy reading! Top 10 blog posts of 2015 1. CRISPR 101: Homology Directed Repair Addgenie Chari...inversions, deletions, and translocations. It also provides researchers with many different ways to control...the right one. In this post, Addgenie Melina Fan guides us through the basics of the validation process...experiment? In this post, Addgenie Chari Cortez guides us through some common controls and why you might...

...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ...of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals...their specificity or from the thymus to the lymph nodes. These methods gave researchers much more information...expressing cells. For example, by harvesting lymph nodes at different times after antigen challenge, scientists... cellular dynamics in lymphoid organs. In lymph nodes, for example, two-photon microscopy allowed scientists...extravasate from the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon...interactions in diverse tissues, including lymph nodes, brain, liver, gut and skin. Two-photon intravital...

...targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...when a high degree of signal to noise is desired. It also provides an excellent additional measure of DNA...

...approximately 20 nm of a specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically...transport from the cell surface. Their toolkit includes a set of plasmids each containing an anti-GFP ...from A. victoria the functionalized nanobodies described here can be used to target any protein fused with...

... 160(1), 339-350 (2015). PubMed. Find the Plasmids @Addgene: Plasmids for CRISPRi are described here...stepwise protocol on designing and using RNA scaffolds: Delebecque, C. J. et al. Designing and using RNA scaffolds...regulators allows a greater degree of control of desired expression levels dependent upon the type of transcriptional...transcriptional regulator or the number of designed scaffold binding sites. With hundreds of characterized...tunability of transcription is possible. From a design perspective, this is appealing since the RNA scaffold...pairing, allowing for rational, straightforward design. Since the technique relies on base pairing, it...and multidirectional multiplex control via scRNA design is readily apparent in metabolic engineering. The...

...Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening using...assign four different barcodes to each of the gRNAs and to embed these barcodes within the gRNA sequences...lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized GFP ...method that makes use of guide RNAs with internal barcodes (iBARs) in their loop region. After infecting ...bioRxiv 2019. https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast...intracellular Ca2+ (bottom panels) equipped with destabilized GFP calcineurin reporter (blue) and stained ...

...and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. Using this...our AAV preparations. We recently published a description of the VGS process and use cases in Human Gene...ligation and tagmentation preparation approaches described previously require a preliminary double stranding... the expression cassette that we want, it also includes a small amount of spurious DNA from the packaging...plasmids. We decided to use this to our advantage by designing a Python script that looks for pre-defined serotype...signature sequences in the data. Unlike previously described thermodynamics-based methods of serotype confirmation...

...heterodimerizes with CIBN upon blue light exposure (see Fig 1*). When the group targeted the enhancer region of ...almost complete desensitization when exposed to continuous bright light. Desensitization is mediated by...discovered Marine, Anion-conducting and Intensely Desensitizing ChRs) differ from currently used ChRs in that...regulation of many genes. A number of groups have designed means of studying the effects of endogenous looping... they call the LADL Anchor. Using specifically designed gRNAs, they can then recruit these LADL Anchors...

...net negative charge with 4 negatively charged and 1 positively charged amino acids. Myc tags are frequently... What are epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein...purification or detection of the tagged protein. These peptides consist of as little as 8 or as many as several...need monoclonal GFP antibodies or guidance on designing a GFP tag Addgene has you covered!   Potential...Protein folding issues, steric hindrances, and destabilization can be brought about by the tag so it’s important...

.... You can email her at [email protected].   References 1. Huang, Ally, et al. "BioBits™ Explorer: A modular...resources to provide them to their students. My desire to create a set of low-cost, easy-to-use educational...protein – you first need the DNA sequence that encodes it. That DNA sequence then needs to be incorporated...one is as cheap as 10 cents, making activities designed with FD-CF reactions affordable to many schools...students. In our BioBits kit for example, we’ve designed FD-CF experiments that result in outputs that ...

...post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple...be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo engineering of oncogenic...ligated into Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt...cell that gets the plasmid contains all of the desired gRNAs. This increases the chance that all the edits...promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target sequences and ...ordered assembly of multiple components into a destination vector. The first step in CRISPR/Cas9 Golden ...Golden Gate multiplexing is to clone the oligonucleotides specifying each gRNA target sequence into distinct...

...class.   Four ways to improve your next manuscript 1. Be consistent with your figures The figures of a ...should also be easily understood and consistent in design and layout. 2. Be concise in your writing  When...

...them you can do today! 10 ways to share your science 1)   If you don’t already make it a habit to talk – ...research, SciFund is really motivated more by the desire to help close the widening gap between science ...

...could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery box...and workflows where CRISPR nuclease activity is desired and time or protocol steps can be saved by flexibly...

...intern to complete and present on.    Figure 1: Addgene's summer interns presenting their capstone...instrument. They are the oldest sibling in a Guatemalan family of Mayan descent.   ...teaching me the basics of physics and sustainable design while allowing me to do my internship in the daytime...

...depending on the virus, cell type, and/or desired event.   Figure 1: Transduction to expression process...retrovirus, lentivirus, adenovirus, or AAV. Then, your desired DNA to be integrated can be cloned into the vector...packaged, when the vector is expressed. Once the design and construction steps are complete, it’s time ...

...heat-fixed smear with the crystal violet stain for 1 minute. Rinse the slide briefly with tap water. Flood...phrase gram negative or positive being used to describe a species of bacteria, but what does it actually...bacteria. In a gram negative bacteria, the alcohol destroys the outer lipid membrane, and the thin inner peptidoglycan... is also useful in a laboratory setting. When designing a plasmid to be inserted into a specific bacterium...

...occur, but good performers move around easily  Table 1: Taking it a Step Further - Big Company v. Small Company...one list can ever encompass them all. We can’t designate non-academic jobs as “alternative” anymore. ...Develop expertise areas quickly Jack/Jane of all trades More sources of training and resources within...

... difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a...without end-point sequencing assays.  This tool was designed by putting together the coding sequence of a mCherry...MYC-CRISPR library targets E-boxes genome-wide. It was designed based on MYC-ChIP-seq data from several MYC-dependent...

...vesicles. Okada CY, Rechsteiner M. Cell. 1982 May;29(1):33-41. PubMed. Protein transduction: unrestricted...transduced using only protein. Read on for a description of the lab’s iTOP protein-only transduction method...transduction by osmocytosis and propanebetaine) to describe this method of protein transduction. Later studies...