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We narrowed to 185 results for: INV-E

Showing: 1 - 20 of 185 results

  1. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...transformed E. coli . Over-agar spreading of antibiotic makes it easy for an investigator to conveniently...stock solution in sterile water (or other antibiotic) E. coli transformed with a plasmid containing the carbenicillin...fully absorb. During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest...transformation protocol. Plate 50 µL of transformed E. coli /rescue media suspension onto the agar and gently...carbenicillin, plated over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on LB-agar...Plate with No Carbenicillin Plate shows a lawn of E. coli and no selection. 150 µL of 0.1 mg/mL Carbenicillin...Carbenicillin plated over-agar Plate shows a lawn of E. coli and no apparent selection. 150 µL of 1 mg/mL ...

  2. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing...secondary structure of the hairpin sequence. Back to Top E. Producing Lentiviral Particles Before this step, ...cells that stably express your shRNA of interest. E.1 Recommended Materials Material Vendor and catalog...information, visit Addgene’s Mammalian RNAi Tools page. E.2 Protocol for Producing Lentiviral Particles This...tube. Incubate for 5 minutes at room temperature. e. Add 80 μL of FuGENE® master mix to each tube from...appropriate puromycin-containing media to cells. Days 3+: e. Examine cells each day and change to fresh puromycin-containing...polybrene. c. Add lentiviral particle solution from step E. For a 6 cm target plate, add between 0.05-1 mL virus...

  3. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    ...Lisowski, L., Alexander, I. E., & Logan, G. J. (2019). A User’s Guide to the Inverted Terminal Repeats of Adeno-Associated...doi.org/10.1089/hgtb.2016.133. PMID: 28967288. Shitik, E. M., Shalik, I. K., & Yudkin, D. V. (2023). AAV- based...elements, but among the most important are the inverted terminal repeats (ITRs). ITRs are the elements...2020). Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene...Merten, O.-W., & Galibert, L. (2017). Impact of Inverted Terminal Repeat Integrity on rAAV8 Production ...2017). Deletion of the B-B’ and C-C’ regions of inverted terminal repeats reduces rAAV productivity but...

  4. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...4 empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample 2 Sample 5 F Perform data analysis...Standard curve: R 2 (coefficient of correlation) ~ 1.0, E (efficiency of PCR) ~100% (90%–110% range is acceptable...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods...

  5. Typing CRISPR Systems

    Type
    Blog Post
    ...Demircioglu, F. E., Moeller, L., Kocalar, S., Oshiro, R., Makarova, K. S., Macrae, R. K., Koonin, E. V., & Zhang...Chylinski, K., Makarova, K. S., Charpentier, E., & Koonin, E. V. (2014). Classification and evolution of... Krajeski, R., Ioannidi, E., Lee, B., Gardner, A., Makarova, K. S., Koonin, E. V., Abudayyeh, O. O., &...W. X., Hunnewell, P., Alfonse, L. E., Carte, J. M., Keston-Smith, E., Sothiselvam, S., Garrity, A. J.,...DNA with Cas3 (pink). Complex shown is from type I-E. Created with BioRender.com.   Type III Type III...tenuous grasp on how type III systems function, a III-E effector was developed, called Cas7-11. Despite being...Cox, D. B. T., Kellner, M. J., Regev, A., Lander, E. S., Voytas, D. F., Ting, A. Y., & Zhang, F. (2017...

  6. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ...found in E. coli have been manipulated for use in molecular biology studies. For example the E. coli biotin...small size, and low cost, it’s understandable why E. coli is the most popular bacterial organism in microbiology...2017).  Escherichia coli. Image from NIAID. E. coli is often a part of the “molecular biologist ...manipulation and their ability to be replicated. Because E. coli are easily propagated and have a generation ...molecular cloning and metabolic engineering.  The E. coli genome has an extensive research history and...2012) and APEX (Rhee et al., 2013). Research using E. coli has the potential to address many areas of microbial...biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these processes...

  7. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

    Type
    Blog Post
    ... out” of ccdB with the investigator’s gene of interest.   CcdB resistant E. coli strains complete...ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded...pKIL19, contained the ccdB gene in frame with an MCS. E. coli that were transformed with the empty vectors...expressing it? The answer lies in special strains of E. coli that tolerate the expression of the toxin gene...ccdA will be present. Most common cloning strains of E. coli do not contain the F plasmid (and are considered...recombinant plasmids. Check out our blog post on Common Lab E. coli strains for more strain info. Additional cloning...Resources on the Addgene Blog Plasmids 101: Common Lab E. coli Strains Plasmids 101: TOPO Cloning Plasmids ...

  8. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...and delight,” the very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight but...Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response to a ~25 mT magnet. Gray...magnetoresponse was clearly visible whether expressed in E. coli, in mammalian cells, or in purified form, and...CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while a magnet is waved under...resources References Duret, G., Polali, S., Anderson, E. D., Bell, A. M., Tzouanas, C. N., Avants, B. W., ...Hayward, R., Rai, A., Lazzari-Dean, J. R., Lefebvre, A. E. Y. T., York, A. G., Ingaramo, M. (2024). Magnetic...doi.org/10.1002/pro.4124.  Kattnig, D. R., Evans, E. W., Déjean, V., Dodson, C. A., Wallace, M. I., Mackenzie...

  9. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...-Cortés, A. E., Walker, M., Kalmbach, B., Lenz, G. H., Morin, E., Nguyen, T. N., Garren, E., Bendrick,...Hess, E. E., Mahoney, J. T., Kojima, Y., Ding, Y., Somasundaram, S., Miller, J. A., Kalmbach, B. E., Radaelli...Taskin, N., Goertsen, D., Shay, T. F., Sullivan, E. E., Huang, S.-F., Ravindra Kumar, S., Arokiaraj, C...10.1038/s41592-020-0799-7 Shay, T. F., Sullivan, E. E., Ding, X., Chen, X., Ravindra Kumar, S., Goertsen...Ibrahim, L. A., Mastro, K. J., Sabri, E., Huang, S., Favuzzi, E., … Dimidschstein, J. (2020). Viral manipulation...., Deverman, B. E., & Gradinaru, V. (2017). Engineered AAVs for efficient noninvasive gene delivery to...//doi.org/10.1038/s41565-023-01419-x Deverman, B. E., Pravdo, P. L., Simpson, B. P., Kumar, S. R., Chan...

  10. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...combination of Cas5, Cas6, Cas7, Cas8, and Cas11. Type I-E Cascades contains all five of these Cas proteins, ... Figure 2: Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex...j.molcel.2018.03.031 Xiao, Y., Luo, M., Dolan, A. E., Liao, M., & Ke, A. (2018). Structure basis for RNA-guided.../doi.org/10.1016/j.ggedit.2022.100013 O’Brien, R. E., Bravo, J. P., Ramos, D., Hibshman, G. N., Wright..., K., Okuzaki, Y., Gee, P., Kunihiro, Y., Sonpho, E., Xu, H., Sasakawa, N., Naito, Y., Nakada, S., Yamamoto...019-13226-x Cameron, P., Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., Donohoue, ...J., Barrangou, R., Brouns, S. J. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F...

  11. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...phage DNA fragments in E. coli Now that random DNA fragments have been cloned into E. coli cells, isolated...fragments so that these fragments can be cloned into E. coli and subsequent proteins screened for cell-binding...digested vector designed for protein overexpression in E. coli, such as pET30a. If working with DNA that is...resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately ...colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3) cells (these have the T7 polymerase...the membranes for 15 min to kill any remaining live E. coli. At this point, it can really help to incubate...conditions. Note: if using fast-growing bacteria like E. coli or Salmonella, or any cells that bind strongly...

  12. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...systems in E. coli. However, some users pointed out that this system did not function properly in E. coli ...in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to take place... can be operated at 37 ℃ for faster growth of its E. coli host, and the addition of a sacB gene renders...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...crRNA arrays targeting multiple different sites in E. coli. pCutamp cures the plasmids by targeting the... Image from Li et al., 2021.   The updates involved the following changes. In pCas9, we 1) replaced...

  13. pSiM24: Simplifying Plant Genetic Engineering

    Type
    Blog Post
    ...Agrobacterium and E. coli. Like plasmids used in other systems, binary vectors are cloned and prepped in E. coli....infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has created a ... contain two origins of replication, one each for E. coli and Agrobacterium, along with a selectable marker... T-DNA borders to 25 bases each. By switching the E. coli origin of replication and codon-optimizing the... (>10 kb.) Since transformation efficiency is inversely proportional to size, and many plant scientists...

  14. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...method in E. coli, Jesse Zalatan’s lab used an approach to identify known proteins involved in the activation... are making it easier than ever to use CRISPR  in E. coli and other bacterial species. Read on to learn...design the first synthetic bacterial CRISPR system in E. coli. The system available from Addgene consists ...targeting the gene of interest and kanamycin resistance E. coli carrying the phage recombineering machinery ... isn’t the primary editing force. In contrast, in E. coli, CRISPR is primarily a means of selection that...protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless editing plasmids Sheng...subsequently cured from the bacteria. Although developed in E. coli, the system was used successfully in Tatumella...

  15. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    ...Fogarty, E. C., Schechter, M. S., Lolans, K., Sheahan, M. L., Veseli, I., Moore, R. M., Kiefl, E., Moody... Chang, E. B., Ruscheweyh, H.-J., Sunagawa, S., Mclellan, S. L., Willis, A. D., Comstock, L. E., & Eren...infect other organisms. If you’ve ever wondered why E. coli is safe to use in the lab but also causes outbreaks...example, Gateway cloning relies on genes found on an E. coli fertility plasmid that also encodes a toxin,...resistance to that toxin, CcdA. Knowing which strains of E. coli contain this F plasmid is critical for successful...This category includes genes that help bacteria invade hosts, including attaching to host cells and evading...

  16. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ...switch in cyanobacteria, synthetic inverters in E. coli, and biosensors in E. coli: Čelešnik, Helena, et al...synthetic biology or protein purification studies in E. coli. 800 and 1900 Series If you’re expressing two... can be used to precisely tune your polycistronic E. coli expression constructs. Some applications of ...2388-2393. Huang, Shijie, Xuechen Zhu, and Charles E. Melançon III. "Detection and Quantification of Ribosome...plasmids in the kit can be used to purify proteins from E. coli, Bacillus subtilis, or Pichia pastoris depending...aggregation propensity in four expression systems: E. coli, wheat germ, HeLa, and Leishmania (Gagoski, ...quickly clone your segments of choice into either an E. coli or a mammalian expression vector in a one-pot...

  17. Developing a cold tolerant E. coli using specialized chaperones

    Type
    Blog Post
    ...suboptimal ones, which could allow even your regular E. coli to thrive in the bitter cold.  Since our ...example would be a transformed mesophilic, for example E. coli, carrying a plasmid expressing a protein of ...ever consider them worth the trouble.  Enter ELSA: E. coli Learning Suboptimal Acclimatisation To resolve...psychrophilic proteins. As the figure below shows, E. coli expressing the chaperones protein resulted in... in higher levels of growth compared to the E. coli strain expressing the empty vector. And thus was born... born ELSA - our happy lil’ cold-tolerant E. coli! Additionally, we’re also working towards widening the...collaborative environment for research.  We’ve also involved ourselves in extensive outreach programs to encourage...

  18. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...Ferenczi, E. A., Ramakrishnan, C., O’Shea, D. J., Prakash, R., Gunaydin, L. A., Hyun, M., Fenno, L. E., Gradinaru.../10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., & Deisseroth, K. (2005)..., J. C., McKnight, D. J., Yoshizawa, S., Kato, H. E., Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific...Berthold, P., Ollig, D., Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated...doi.org/10.1073/pnas.1936192100 Tichy, A.-M., Gerrard, E. J., Sexton, P. M., & Janovjak, H. (2019). Light-activated...Ahlbeck, J., Augustin, S., Sauter, K., Papagiakoumou, E., Gottschalk, A., Soba, P., Emiliani, V., Engel, A...

  19. Using Ultrasound to Image Bacteria in vivo: Acoustic Reporter Genes

    Type
    Blog Post
    ... expressing E. coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain...created two constructs optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 ...with 20 hours of growth following acoustic collapse E. coli re-express arg1 gas vesicles and restore their...arg1 vesicles. Following collapse, vesicles are invisible to ultrasound, which allows for differentiation...Shapiro, M.G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts....

  20. Three Tips for Preventing Viral Plasmid Recombination in Your Samples

    Type
    Blog Post
    ...failed experiments. Pick the right strain While common E. coli strains like DH5-alpha are recA-, some strains...Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce the rate of recombination in samples....common gene mutations in our Plasmids 101: Common Lab E. coli Strains blog post. Check yourself before you...the round tan colonies that look similar to normal E. coli cultures contain the recombined vector backbone...viral vector protocols Learn about the common lab E. coli strains Find tips for viral transduction  ...most frequent problems that I help troubleshoot involves viral vectors and plasmids with repeating sequences...
Showing: 1 - 20 of 185 results