...that are built into how something functions, were designed for this purpose. One such control is dividing...

... and to work in a variety of applications. The desired specificity of your secondary antibody depends ...

... Parts. This library delivers 2 randomly paired guides per construct and allows a reduced scale in screens...

...uncomfortable places, from sticky ethical dilemmas to dark episodes in the history of eugenics and research misconduct...

...materials required for each [1, 2]. This protocol [3] provides, in exquisite detail, the various steps required...

...communication and learning to work with people despite the distance improved my verbal and written communication...

...Derek Jacoby from Makerspace Victoria, CA Over the decades science has become increasingly restricted to academic...

...familiar with the minute details of your work. Describe the problem by stating the facts and explain how...

...Vector Guides Virus Blog Posts Addgene Protocols Viral Service Introduction This protocol describes how ...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells...type will be able to grow under the conditions described in this protocol. Conditioned medium (details ... specific conditions used. Each 96-well plate described in this protocol could, in theory, give rise to...protocol was performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise...from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved vectors and genome-wide libraries ...

...experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers... Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested leader...primer is shown here. Note: Use web-based primer design software to ensure a melting temperature between...nucleotide dGTP in the reaction (exclude all other nucleotides from standard polymerase protocol), causing the...manufacturer. It is very important to remove all free nucleotides from your PCR product before proceeding, as they...

...and could be contaminated.Learn how Addgene uses barcodes to track samples Keep track of passage numbers...

...investigators to identify specific clones with desirable properties, either initially, or using secondary...

...(the Gal4/UAS system), and the third post will describe how you can make your own mutant flies. Find Drosophila...

...Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here Additional...

...specific details about the individuals, each example provides a scenario where both people get something valuable...

...Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis. Nature Plants,...

... (about three to four days), this second round provides the added benefit of producing a stock P1 virus...

...Maria were encouraged by previous reports that described magnetic responses of cryptochrome proteins or...

...This will make it difficult to properly gate your desired cell population. By compensating between fluorescent...

...components into mammalian cell lines is quite broad and includes several different methods, so we've summarized...

... to a new plate containing the mastermix which includes primers and a ddPCR supermix. Note that the supermix...

...minigenome system Minigenome systems, such as the one described here, are extremely useful research tools. In ...

...activation. Including a prey-only control also provides a baseline level of reporter activation. Vary ...

...that extra two miles which is way more fun. And despite those who may wish to tell you so – towel corners...

...plasmid of interest. For this example, we will describe how to copy a cDNA from one vector into a new ...(YGOI) for ligation into a recipient plasmid. Designing Primers for PCR Based Cloning The basic PCR primers...that: Do not cut within your insert. Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement...

...This protocol describes the basic steps for lysing cells, determining total protein concentration in ...specific to the protein of interest. This protocol describes the basic steps for lysing cells, determining ...continue running the gel until you have obtained the desired separation. Gently open the gel with the spatula...Device. Close the lid of the device. Select the desired method and make sure the parameters are correct... incubation Dilute the primary antibody to the desired concentration in blocking buffer. Pro-Tip The optimal...peroxidase-conjugated secondary antibody to the desired concentration in blocking buffer. Pro-Tip The optimal...

...collaborators than you realize. Being at a university provides the perfect platform for practicing initiative...

... this re-inversion issue. It became possible to design FLEx vectors (Flip-excision vectors) that only ...

... plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and subsequent...

...an abasic site. Like in most other base editor designs, the Cas9 nicks the non-edited strand to encourage...

...will identify a ‘scientific mentor’: someone who provides career advice and offers direction on your scientific...

...primer should be a minimum of 50 nucleotides and a maximum of 300 nucleotides from your target. Are there ...Analysis of a Plasmid You may also like... How to Design a Primer Diagnostic Restriction Digest Introduction...If you require additional sequencing and need to design a custom primer, Addgene recommends using Addgene's...'s sequencing results as a reference for primer design. A good sequencing reaction will produce between...

... of bacteria or fungi. There are disinfectants designed to be used in water baths with instructions on...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...Set up your water bath so that it will reach the desired temperature by the time you need it for your experiment...water bath to prevent evaporation and maintain the desired temperature. This also helps the water bath heat...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody...antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells ... per well. Allow the HeLa cells to grow to the desired density before labeling. Section 2: Fixing and ... container. Dilute the primary antibody to the desired concentration in antibody dilution buffer. Pro-...fluorescently-labeled secondary antibody to the desired concentration in antibody dilution buffer. Pro-...

...if I had planned my day extensively, my lack of desire to go sit in the tissue culture hood may have prevented...

...difficult and time consuming. GAL4/UAS was first described in 1993 by Norbert Perrimon’s lab and has been...

...even better. The Thomas Kilmann Conflict Model describes 5 basic approaches to conflict resolution based...

...Biol 24, 758-766 e753 (2017). Baloban, M. et al. Designing brighter near-infrared fluorescent proteins: insights...

...with a site mutation (R11G). This site mutation destabilizes the ligase, allowing for active biotin molecules...

...Collection of guides that describe how popular scientific techniques work... Science Guides Addgene has created... created the following guides to help you start learning about various scientific concepts related to ...to our plasmid collection. Find links to the guides below. Antibodies Chemogenetics CRISPR Optogenetics... sets. Read More Viral Vectors Find educational guides to learn more about the different types of viruses...

... it includes labs that work with agents associated with diseases in healthy humans. BSL-2 includes all...There are four biosafety levels. This protocol provides information for both biosafety level 1 (BSL-1)...important! Disclaimer: The SOP presented here has been designed by the Addgene nonprofit plasmid repository and...

...addgene.org with any questions. Protocols Video protocol guides for standard laboratory procedures. How-To Videos...Protocols (Link opens in a new window) Video Link Description Related Page Making LB Agar Plates Create plates...Videos (Link opens in a new window) Video Link Description Related Page MTA Guide - Requests Your guide ...Videos (Link opens in a new window) Video Link Description Jessica Welch, PhD In this first installment ...

...Cell type-specific delivery by modular envelope design. Nature Communications, 14(1), Article 1. https...

...magnifies it 10 times, and so on. The ocular lens also provides magnification and the power should be provided...provided on the microscope; often this lens provides 10x magnification. To determine the final magnification...that object! Equipment Light Microscope Prepared Slides (or other sample that can fit on a microscope stage...eyepiece so that you can see your image through both sides. Once you are happy with the lighting, use the coarse...

...be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly...Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and ...Always Run to Red. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove...-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that has UV light,...

...may also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction...recheck the pH to prevent over or undershooting the desired pH. Allow the solution to mix for 10 min and then...2-3 minutes for cells to detach. Gently tap the sides of the CS2 to help detach the cells, add 200 mL ...for ~72 h. Harvest cells and media by tapping the sides of the CS5. Cells should detach easily. Transfer...

...library during amplification. This protocol is designed to be as general as possible but note that individual...activating pulse and follow all specifications described in the equipment manual. Immediately add 1 mL ...µl aliquots of cell/DNA mixture used (8 in the described protocol) using a new vented falcon tube for every...than Nanodrop, although most protocols have been designed with Nanodrop as the quantification method. If...

...Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service Introduction This protocol describes a general...recheck the pH to prevent over or undershooting the desired pH. Allow the solution to mix for 10 min and then...

...phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically... likely to ligate to itself rather than to the desired insert. If you are in this situation, it is important...reactions. Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it is a good idea to ...

...maximum possible setting on the pipette, set the desired volume of the liquid on the pipette. Depending ...of the pipette. Select the pipette tip that is designed to fit your pipette. To load a tip onto a pipette...from the container making sure not to touch the sides of the container with the pipette. Discard the pipette...