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We narrowed to 634 results for: des.2

Showing: 561 - 580 of 634 results

  1. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression...and some of the most popular commercial strains designed to avoid them. Why do I need an expression strain...protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce proteolysis of the...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...and pLysE—the difference being that the latter provides tighter control of basal expression. What if I...don't see protein overexpression? The strains described above should generate sufficient expression levels...you’ve tried a common strain and don’t get the desired level (or any) protein expression? Low expression...

  2. 5 Reasons to Use Reddit for Science Communication

    Type
    Blog Post
    ...community is active, engaged, and has great questions! 2. The r/Science subreddit is well moderated. One of...They also host AMA forums where users can ask designated scientists anything about a particular topic....calendar invite over to the r/Science team with a description of our AMA and Joanne’s preferred time to start...was writing all day and straight on through her designated two hours for answering questions. She was able...toxic advisors, fruitless job searches, and even despair at having chosen an academic past. I hope I was...receive via email. The cool features are too many to describe here, but I have over 2,000 notes which Evernote...

  3. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ... PMID: 9537413. PubMed Central PMCID: PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating...human cells (Kim et al, 2016). The Waldman lab has designed their AAV tagging vectors (pAAV-SEPT-Acceptor ...vectors for your specific gene, the lab recommends designing homology arms with the modification of interest...combining the AAV and CRISPR-based approaches is described in several recent reviews (Howes and Schofield...

  4. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ...Nature 339 (1989). 487–488. PubMed PMID: 2725683. 2. Miller, C.J., Kassem H.S., Pepper S.D., Hey Y., Ward...best to start from scratch. Obtain a vial of the desired cell line from a reputable distributor such as ...extreme changes in osmotic pressure and pH can destabilize the viral envelope. One drawback of the multiple...

  5. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...AAV variants for widespread gene transfer to the adult brain. Nature Biotechnology, 34(2), 204–209. https...start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid, while... highlighted below the table.     Figure 2: This decision tree can help guide your choice on ...expression to specific tissues and cell types. If desired, spatiotemporal control of transgene expression...systemic AAVs to find commonly used vector cargo designs and administration doses. Note: If capsid immunogenicity... Table 1 gives a brief overview of published descriptions of commonly used or emerging capsids. Selected..., B.E., Gradinaru V. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nature...

  8. Protocol - How to Design Primers

    Type
    Protocol
    ...-24 bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer... Protocols Designing Primers How to Design a Primer You may also like...Tips on designing primers for PCR Protocols...Cloning by PCR Agarose Gel Electrophoresis Primer Design for PCR Oligonucleotide primers are necessary when... when running a PCR reaction. One needs to design primers that are complementary to the template region.... They are synthesized chemically by joining nucleotides together. One must selectively block and unblock... the primer usually has stretches of several nucleotides. Also, both of the 3’ ends of the hybridized ...

  9. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ... Methods 2009; 6, 343-345. PubMed PMID: 19363495. 2. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey... friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “...assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. ... Both Snapgene and NEB have tools that help you design primers for PCR amplification of fragments to incorporate...demonstration of how to use Snapgene’s program to design primers for Gibson Assembly. For a simple example...without scars as depicted below. First, you need to design primers to amplify the two fragments while also...the process works best with fragments over 200 nucleotides. This may be because the exonuclease could chew...

  10. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ... more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...Addgene’s catalog is a new class of sensors for neuropeptides and neurotransmitters. Motivated to develop ...GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost A to Z! - from ATP...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific... tool for monitoring RTK signaling.  Figure 4: Design of pYtag fluorescent biosensors for monitoring ...iGECInano plasmids here!   Matlashov ME, et al. Design and Initial Characterization of a Small Near-Infrared...intermediate affinities from 40 to 140 mM. These new designs greatly broaden the options for potassium biosensors...

  11. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ... infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs to target the...the virus doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1,... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target the 5’ long terminal ...could be used to combat this problem, just as ART includes multiple drugs to lower the odds of developing...strategy may be a better option than direct cleavage. Despite the potential difficulties in translating these...

  12. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ..., whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which is...any DNA sequence in the way that they like, the design and engineering of fusion proteins is relatively...fusion proteins and highlight some aspects of their design.  Protein size and shape matters The green fluorescent...necessary for post-translational modifications. Despite these potential issues, GFP has been used successfully...consist of random amino acid sequences or (ii) designed to form an inert, unstructured peptide and therefore...C-terminus of the fluorescent protein. I prefer to describe the different parts of the fusion from N- to C-terminus...2011).  Although structural information may guide design, it is advisable to generate multiple constructs...

  13. A Control for All Seasons

    Type
    Blog Post
    ...as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should include...controls and be able to use this information to design thoughtfully and thoroughly controlled experiments...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the...the Fc, allowing you to detect this undesirable interaction. When non-specific interactions are suspected...nonspecifically with positively charged groups, such as aldehydes in fixed samples. To address this, block positively...

  14. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ...two visible blue or magenta chromoproteins (Figure 2) (Liljeruhm et al., 2018). This system is a nice confirmation...scarless mutations, ones that don’t leave any undesired scars in the chromosome. These methods roughly... recombination enzymes can also accomplish the desired genomic modification. This technique is frequently...resolution step (“double crossover”) which leads to the desired mutation and excision of the vector backbone. This...the pDS132 MCS with a versatile, synthetic MCS (designed with the help of the DNA tuner program) (Latynski...purified amplicons upstream and downstream of the desired deletion directly into cut vector with excellent... of interest, from the day primers arrive, the desired chromosomal modification can be made in 5 days....

  15. Chemogenetics Guide

    Type
    Guide
    ...their effect, and outcome in neurons. Table 2: Descriptions of PSAMs and their activity in neurons PSAM...muscarinic receptor and has not been used in vivo. Figure 2: DREADDs, their ligands, and signaling properties.... Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...hM1Dq hM5Dq Human M3 muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al., 2007 rM3D...FRET-modified bioluminescence. Neurophotonics 11 (2), 021005. https://doi.org/10.1117/1.NPh.11.2.021005... Guettier, J. M., Gautam, D., Scarselli, M., Ruiz de Azua, I., Li, J. H., Rosemond, E., Ma, X., Gonzalez...improved coupling efficiencies. Neurophotonics, 11 (2), 024208. https://doi.org/10.1117/1.NPh.11.2.024208...

  16. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...PCR tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ... reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ...normally sterile dH 2 O. To make a 100uM stock of any primer, add a number of µl of dH 2 O equal to the number...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can ...work adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations...Sterile dH 2 O 0.2 μL Taq DNA Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer Design and PCR...PCR to work efficiently. Taq Buffer with MgCl 2 : Provides an optimal and stable chemical environment for...

  17. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...worn outside of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names and contact...protocol provides information for both biosafety level 1 (BSL-1) and biosafety level 2 (BSL-2). The purpose...in addition to BSL-2 guidelines below, including PPE protocols . Working in a BSL-2 laboratory requires...Staphylococcus aureus or Vibrio cholerae . BSL-2 includes all of the precautions needed in BSL-1, along...pouring down the drain. Solid BSL-2 waste can be collected in designated biohazardous waste containers that... steps to ensure you are working in BSL-1 and BSL-2 labs safely. Protocols... Biosafety Levels One and Two (BSL-1 and BSL-2) Intro to the Lab Bench Check out more protocols and videos...

  18. Modular Cloning Guide

    Type
    Guide
    ...into a Level 2 vector, forming a functional genetic circuit. Level 2 vectors are often designed with flexibility...of three sets of cloning vectors (Level 0, 1, and 2) which can be used in successive assembly steps. Before... iterations of assembly. Combining multiple Level 2 vectors permits the creation of even more complex ... 16 RBS strength variants, 8 tag-compatible RBSs, 2 secretion tags, 6 CDSs, 9 terminators, 4 nonfunctional...of multigene constructs. PLoS One . 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PMID: ...Yamamoto Nine pFUS array vectors and destination vectors designed for use with the Golden Gate TALEN and...MoClo kits in our collection. Kit name Type PI Description MoClo Toolkit Plant Expression Sylvestre Marillonnet...

  19. CRISPR Guide

    Type
    Guide
    ...M. M., Semenova, E., Severinov, K., De Vos, W. M., Dame, R. T., De Vries, R., Brouns, S. J., & Van Der...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Browse Plasmids: Double-Strand Break (Cut) Figure 2: Overview of the NHEJ repair mechanism. Multiplex ...systems enable researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single...Cas9 is included in the gRNA-containing plasmid, or 2-vector systems, in which Cas9 must be delivered separately...your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either co-infect with...the presence of infectious organisms (like SARS-CoV-2 ) and genetic mutations. Similar to Cas9 and Cas12...

  20. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...materials D.2 Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol...Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty...VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing Oligos...oligo 5 μL Reverse oligo 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick...buffer for EcoRI 1 μL EcoRI 14 μL ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting... For a standard T4 ligation, mix: 2 μL annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning ...
Showing: 561 - 580 of 634 results