...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...

...needs. For instance, I had one colleague who preferred detailed instructions and regular check-ins, which ... made similar transitions can provide you with insights, lessons from their experiences, and practical...

...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. Created with...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...

...probasin Mammalian Green 8405 p240 pPr-CREM/Ins CREM with insulators probasin Mammalian Green 8406 p252 pPr-CREM... p255 pPr-CREM/Ins/CMV-STOP-luc CREM and CMV-STOP-luc cassette, separated by insulator probasin Mammalian...coexpression Retroviral Littman 24333 ins-cre_cryaa-Venus Cre-Venus fusion insulin Zebrafish Stainier 24593 AAV-pgk-Cre...for inserting promoter none Mammalian Heller 112616 pmTHC TFP and Cre-ERT2 with MCS for inserting promoter...recombinases that are less toxic in Drosophila hsp70 Insect Rubin 32606 Ttr:Cre Cre expressed in visceral endoderm...CMV AAV Gage 49111 pEMS1980 iCre with MCS for inserting promoter none AAV Simpson 49455 pSH62-EBD Cre-...troponin T AAV Pu 70120 pEMS2159 iCre with MCS for inserting promoter, WPRE none AAV Simpson 72255 pCDH-CB-...

...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 ...

..., 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. ...your donor ssDNA. Here, we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et...

...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...

...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...

...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...

...Appropriate restriction enzyme (see manufacturer's instructions for proper ammount) Approrpriate restriction...restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips Procedure...an appropriate reaction buffer by reading the instructions for your enzyme. Pro-Tip If you are conducting...for 1 hour. Always follow the manufacturer’s instructions. Pro-Tip Depending on the application and the... the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours ... involve incubating the reaction at 70 °C for 15 mins, or purifying the DNA via a purification kit, such... cleanup kit . See the enzyme manufacturer's instructions for more details. To visualize the results of...

...competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock...many genetic modifications to create bacterial strains that can be more easily transformed and that will...efficiency, we recommend that you follow the instructions that came with your competent cells. Pro-Tips...out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate...be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. ... the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing ...bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will...

...weight proteins and higher percentage acrylamide when immunoblotting low molecular weight proteins. This...Transfer for 5–6 min for proteins <30 kDa. Transfer for 8–10 min for proteins >150 kDa. Select Start Run...Introduction Western blot is a technique used to separate proteins by size followed by detection using antibodies...Incubate with secondary antibody Video Watch this instructional video to learn how to use western blotting to... Before Starting Refer to the manufacturer's instructions for additional information specific to your ... of interest. RIPA buffer is suitable for most proteins but more stringent buffers and a sonication step...step may be required for hard to extract proteins such as those in the nucleus. Incubate on ice for 30...

...poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely ... a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that...flask. See TAE Recipe . Pro-Tip TBE can be used instead of TAE, labs usually use one or the other, but ...comfortably keep your hand on the flask), about 5 mins. Optional : Add ethidium bromide (EtBr) to a final... need to soak the gel in EtBr solution and then rinse it in water before you can image the gel. Pour the...you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the...causing it settle to the bottom of the gel well, instead of diffusing in the buffer. Once solidified, place...