...be used in a lab? Here we’ll be talking about the ins and outs of this classification system, including...minute. Rinse the slide briefly with tap water. Flood the slide with Gram's iodine solution. Rinse briefly...immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide ...specific procedural instructions, including timing, preparing the smear, and mixing the stains and solutions...biologist named Christian Gram realized that certain stains were retained only in some species of bacteria....negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com/photos/occbio/7092385559...laboratory setting. When designing a plasmid to be inserted into a specific bacterium, it can be important...

...screen vs. a selection and we are here to discuss the ins and outs of both approaches. Screens and selections...players in a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that ...specifically looking at cells that can survive. Instead, all manipulations are assessed, regardless of ...considering. RNAi screens are similar to CRISPR, but instead of knocking out the genes of interest (or the whole...

...One-on-Ones: Managers should set up regular check-ins with their employees. 15-30 minutes weekly or monthly...who could potentially be impacted by a project. Instead, invite the experts on the topic at hand. Invite... people of an updated protocol), send an email instead. The 3 A's - Agendas, Action items, and Accountability...parts where people had questions or suggestions, instead of reading the whole thing together. How long...FInd Tips on Picking a Research Project Read Other Inside Addgene Posts Resources on the Addgene Website...

...developed new CRISPR-based methods for various knock-ins, discussed in our CRISPR 101 eBook. It’s hard to...targeting vector designed to insert a resistance gene. The vector contains a neomycin resistance gene (...sequence using a specially designed vector that contains sequence homology to the locus of interest. To...negative selection marker HSV-tk is used to select against random recombinants.   Design your targeting...recombination, the negative selection marker selects against improper, random recombination into a different...After “floxing” the resistance gene through the insertion of flanking LoxP sequences, you can remove the...Figure 2: A knockout targeting vector designed to insert and later remove a resistance gene. In this construct...

...homology directed repair to make precise edits or knock-ins. Editing efficiency decreases drastically once your...away from the insertion site, so HDR will be difficult if there’s no PAM near your insertion site. Base ...upstream of the PAM. Even if there’s a PAM near your insertion site, the corresponding gRNA might be undesirable...multiple PAMs should have a fitness advantage over proteins with single PAM recognition. After 24 days of...were capable of cleavage, they tested the clones against an NNN PAM library in bacteria where cleavage leads...

... your best self to the training experience.  The ins and outs of being a trainee There is a lot of advice...technical mentorship from a ‘bench mentor’. In some instances, the bench mentor is also the scientific mentor...different, so let your mentor know what yours is! For instance, some people like to ask questions throughout ... clarification at first, but in the end everyone wins. Your mentor learns where you are at and the level...

..., the efficiency of Easi-CRISPR for simple knock-ins and floxed models varies from 8.5-100% of pups born...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.” Genome Biol. ...standard laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria....remain more difficult to make. For example, large insertions, like a fluorescent protein knock-in, suffer ...intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand breaks ...other undesired outcomes, including one loxP site insertion and one NHEJ-repaired indel, often occur. Single-stranded...your donor ssDNA. Here, we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et...

...their controls!). This blog post will discuss the ins and outs of controls for biological experiments, ... experimental (?) groups were run on a western against a protein standard (P) to check for tau molecular...antibody specificity is to test the primary antibody against wild-type and knockout samples. In this test, wild-type... each experimental run as long as the protocol remains the same.    Figure 2: Microscopy-based ...positively charged groups with generally sticky proteins such as bovine serum albumin. Secondary antibodies...all secondary antibodies are included. If, for instance, you omitted a mouse IgG2a isotype primary antibody... cells only (for example, propidium iodide only stains dead cells). Compensation controls address the ...

...GFP CMV Parkinson's L. Miguel Martins 14122 pcDNA3-HtrA2-EGFP S306A HTRA2 GFP CMV Parkinson's L. Miguel...Miguel Martins 14124 pcDNA3-delta133HtrA-EGFP HTRA2 GFP CMV Parkinson's L. Miguel Martins 14126 pET20b-delta133HtrA2...Miguel Martins 15939 pcDNA3-HtrA2-FLAG S142D HTRA2 Flag CMV Parkinson's L. Miguel Martins 15940 pcDNA3-HtrA2...Flag CMV Parkinson's L. Miguel Martins 16153 pET20b-delta133HtrA2 S142A HTRA2 His T7 Parkinson's L. Miguel...Miguel Martins 16154 pET20b-delta133HtrA2 S142D HTRA2 His T7 Parkinson's L. Miguel Martins 16155 pET20b-...His T7 Parkinson's L. Miguel Martins 16156 pET20b-delta133HtrA2 S400D HTRA2 His T7 Parkinson's L. Miguel...Miguel Martins 16157 pET20b-delta133HtrA2 A141S HTRA2 His T7 Parkinson's L. Miguel Martins 16158 pET20b-...

...of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used...standard cloning (where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will ...vector and insert DNA to use. Combine the following in a PCR or Eppendorf tube: Vector DNA Insert DNA Ligase...transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector ratio is usually sufficient...construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly...join the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and ...called "sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends are...

...choose enzymes that: Flank your insert, but do not cut within your insert Are in the desired location in...commonly used. Follow the manufacturer’s instructions. Isolate your insert and vector by gel purification Run...DNA . Ligate your insert into your vector Conduct a DNA Ligation to fuse your insert to your recipient...plasmid to insert. It is also important to set up negative controls in parallel. For instance, a ligation...cut elsewhere on the plasmid Will result in your insert being in the correct orientation in the recipient...of your gene!) Are in frame with tags or fusion proteins in the recipient plasmid (if you are creating ...specifically designed test digest later to verify that the insert was cloned in the correct orientation. If you cannot...

...the gel can indicate if the plasmid contains the expected size insert. By selecting the appropriate enzyme...manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert and ...plasmid. Verifying Insert Orientation by Restriction Digest If you clone an insert into a vector by a ...orientation of the insert. By choosing an enzyme (or enzymes) that cuts within the insert, but significantly...diagnostic digest to verify plasmid size, verify insert orientation, and more. Protocols...restrictions sites. Often, the size of the plasmid insert and vector backbone are known and thus this technique...the entire construct or excise some or all of an insert from it. Before beginning your diagnostic digest...

...four protein chains: two large heavy chains and two small light chains. These four domains come together...their monovalent nature. Darpins - Darpins are genetically engineered small proteins derived from ankyrin ...ankyrin repeat proteins. These are antibody mimics instead of modified antibodies and are not closely related...synthetic binding proteins which use the Fibronectin type III domain as their scaffolding instead of the antibody...Denature proteins, using heat and/or chemicals, and run on an SDS-PAGE gel to separate proteins by size...your antibody’s specificity. A blank (contains water or buffer instead of a biological sample), which captures... lab to specifically and tightly bind to other proteins. Antibodies, produced as part of the immune system...

...commonly used. Follow the manufacturer’s instructions. Isolate Your Insert and Vector by Gel Purification Run...DNA . Ligate Your Insert Into Your Vector Conduct a DNA Ligation to fuse your insert to your recipient...plasmid to insert. It is also important to set up negative controls in parallel. For instance, a ligation... to choose enzymes that: Do not cut within your insert. Are in the desired location in your recipient ...our cDNA into the recipient plasmid. Remember to insert your DNA in the correct orientation in the recipient...Ppurify the PCR Product Run PCR to amplify your insert DNA. It is important to use a high fidelity taq...of DNA). Once you have cut out and purified your insert and vector bands away from the gel, it is important...

...of small proteins called Acr (Anti-CRISPR) proteins to control CRISPR activity. Acr proteins are found...(deletions or small insertions) or (B) span the genome-insert boundary (insertions only). The PCR product...of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain...effector proteins (e.g. SunTag system , panel B ); dCas9 fused to several different activation domains in series...scaffold RNA that contains the gRNA and an RNA hairpin to recruit activation proteins. CRISPRi has also...recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize targeted genomic...inhibition by anti-CRISPR proteins. 2015. Bondy-Denomy J, Garcia B, Strum S, Du M, Rollins MF, Hidalgo-Reyes ...

...white precipitate will be formed which contains the bacterial proteins and genomic DNA. Incubate tube on ice...tube for 5 min at 12,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles...min at 4°C. Notes: Pellet contains the precipitated DNA. Supernatant contains residues, salts, and water...If you will be using a kit, follow the kit's instructions. If you want to perform plasmid purification... This step brings the pH back down causing the proteins and genomic DNA to precipitate, while leaving ... smaller plasmids free in solution. Pellet the proteins and genomic DNA by centrifugation, and remove ...that has been purified away from the bacterial proteins and genomic DNA. Depending on the method used,...

...your institution’s Bio-Safety office to receive permission and institution-specific instructions. You ...bacteria D. Screening for Inserts D.1 Recommended materials D.2 Screening for inserts E. Producing Lentiviral...Consortium has built a library of shRNAs directed against 15,000 human and 15,000 mouse genes. Addgene is...selection. Figure 1 : Map of pLKO.1 containing an shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb... Negative control vector containing non-hairpin insert. pLKO.1 – scramble shRNA Negative control vector...targets for your gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection...Additional design rules can be found at the Broad Institute's TRC shRNA Design Process . To minimize degradation...

...Firefly Insertion of 5' promoter/enhancer regions Bert Vogelstein 12178 pIS0 Firefly Insertion of target... Renilla Insertion of target sequence in the 3' UTR David Bartel 12177 pIS2 Renilla Insertion of target...Firefly Insertion of 5' promoter/enhancer regions Joshua Mendell 60323 pGL4.23-GW Firefly Insertion of 5'...improved normalization. John Atkins 167576 mpCHECK2 Firefly/Renilla Insertion of 3' UTR sequences downstream...construct that contains your regulatory element of interest. ID Plasmid Description Gene/Insert PI Return ...Plasmid Guide You may also like... Fluorescent Proteins Blog: Luciferase 101 Blog: Technologies Enabled...organisms, ranging from bacteria to aquatic animals to insects. Luciferase catalyzes the oxidation of a substrate...

...Plasmids 101: E. coli strains for protein expression Plasmids 101: Common Lab E. coli Strains Why Bacteria? E...Photoswitchable fluorescent proteins Various PIs Fluorescent protein fusion: contains empty vectors with N and...Expression: contains mEos2 with an N-terminal His tag. Photoactivable fluorescent proteins like this one... 3OC6HSL (Quorum sensing molecule) Cynthia Collins Contains luciferase inducible by the quorum sensing...factor bind to a particular sequence? Do these two proteins interact? What happens when I overexpress this...From recombineering to CRISPR , this collection contains a variety of tools to help you harness bacterial...binding sites Philippe Cluzel RNA localization: Contains a 2x array of the binding sequence for RNA-binding...

... E MGC4638 INS insulin ILPR, IRDN INS-IGF2 INS-IGF2 readthrough transcript - INSL3 insulin-like 3 (Leydig...MGC119819, RLF, RLNL INSL4 insulin-like 4 (placenta) EPIL, PLACENTIN INSL5 insulin-like 5 MGC126695, MGC126697...MGC126697, PRO182, UNQ156 INSL6 insulin-like 6 RIF1 INSR insulin receptor CD220, HHF5 JAG1 jagged 1 (Alagille...communication between cells of the immune system. Interleukins Interleukins are cytokines that are secreted by immune... omega 1 - IGF1 insulin-like growth factor 1 (somatomedin C) IGF1A, IGFI IGF1R insulin-like growth factor...MGC18216 IGF2 insulin-like growth factor 2 (somatomedin A) C11orf43, FLJ22066, FLJ44734, INSIGF, pp9974 IGF2R...bA12D24.1.1, bA12D24.1.2 RLN3 relaxin 3 H3, RXN3, ZINS4, insl7 RNASE2 ribonuclease, RNase A family, 2 (liver...