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We narrowed to 1,050 results for: ins;

Showing: 61 - 80 of 1050 results

  1. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    ...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...

  2. Brzezinski Lab CRISPR Collection

    Type
    Collection
    ...Collections Brzezinski Lab CRISPR Collection Brzezinski Lab CRISPR Collection The Brzezinski lab investigates...empty backbones that utilize Golden Gate cloning to insert the desired guide RNA sequence by digestion of ...utilizing one of the dual guide shuttle plasmids to insert a second guide within a U6-guide cassette. This...Cloning Protocol (PDF, 237 KB) Dual guide cloning: Brzezinski Lab Dual Guide Protocol (PDF, 990 KB) Plasmid...R., Abraham, J., Hensley, A., Jones, K. L., & Brzezinski, J. A. (2021). Initiation of Otx2 expression ...Goodson, N. B., Kaufman, M. A., Park, K. U., & Brzezinski, J. A. (2020). Simultaneous deletion of Prdm1...

  3. Allen Institute for Cell Science Plasmid Collection

    Type
    Collection
    ...Allen Institute for Cell Science has deposited plasmids to generate fluorescently tagged proteins of key...Depositor Collections Allen Institute for Cell Science Plasmid Collection Allen Institute for Cell Science Plasmid...Subcellular Localization Plasmids Protocols The Allen Institute for Cell Science has produced the first publicly... will be able to generate fluorescently tagged proteins of key cellular structures in human cell lines...additional cell line information from the Allen Institute for Cell Science data portal (Link opens in a ...cellular structures. Addgene ID Plasmid Allen Institute ID Tag Protein Structure 87420 PXN-EGFP AICSDP...114406 SMC1A-mEGFP AICSDP-57 mEGFP SMC protein 1A Cohesins 114407 NUP153-mEGFP AICSDP-50 mEGFP Nucleoporin...

  4. Allen Institute for Brain Science AAV Enhancer Collection

    Type
    Collection
    ... Allen Institute for Brain Science AAV Enhancer Collection You May Also Like... Allen Institute for Brain... from Allen Institute's Genetic Tools Atlas (Link opens in a new window) . Allen Institute for Brain Science...Enhancer Plasmids The Allen Institute for Brain Science Enhancer Collection contains best-in-class AAV plasmids...cell populations were tested in mouse brains. This collection contains plasmids for cell populations in the...created by the Allen Institute for Brain Science. Depositor... Depositor Collections Allen Institute for Brain Science...drive cell type-specific expression. The Allen Institute for Brain Sciences has screened thousands of enhancers...

  5. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...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...

  6. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    ...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...

  7. How to Be an Excellent Trainee

    Type
    Blog Post
    ... 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...

  8. A Control for All Seasons

    Type
    Blog Post
    ...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 ...

  9. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ..., 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...

  10. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...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...

  11. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...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...

  12. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...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...

  13. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...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... Purify 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...

  14. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...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,...

  15. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...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...

  16. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...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...

  17. Protocol - Bacterial Transformation

    Type
    Protocol
    ...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...

  18. Western Blot

    Type
    Protocol
    ...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...

  19. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...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...

  20. Ligation Independent Cloning

    Type
    Protocol
    ...: Amplify Insert by PCR Perform PCR amplification of your insert following the instructions provided by...Set up a vector only control with water instead of the insert. Incubate the annealing reaction for 5 minutes...digestion and PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations...the free 3' ends, following the manufacturer's instructions. In this case, we will include the free nucleotide...accomplished by gel purification . Step 5: Create Insert Overhangs Treat your purified PCR product with ...Anneal and Transform Mix your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20...concentration fixed across multiple concentrations of insert, thereby increasing your chances of success. Set...
Showing: 61 - 80 of 1050 results