...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system: a vector...protein purification. For a more thorough introduction to pCri, please see the original publication cited...expression and purification: a comprehensive review of affinity tags and microbial applications." Biotechnology... Protein purification can be one of the most stressful lab activities. Working with proteins requires...interest can save a lot of time later. Protein purification specialists recommend doing some research, notably...Gomis-Rüth, aims to take the guesswork out of protein purification through simple and efficient multiplexing. pCri...sites should one need to remove the tag post-purification. A sample of pCri constructs: pCri-1a, -4a ...

...into more specialized cells with specific functions [1]. In many tissues, stem cells serve to replenish/replace...damaged cells that no longer function adequately [1]. Stem cells’ ability to differentiate into multiple...://www.linkedin.com/in/kmukherjeephd/. References 1. Dittrich, R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive...The potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C....preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1): p. 46-53. PubMed PMID: 17989069. 8. Takahashi, K...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...Unipotency: As the name suggests, cells under this category can differentiate into only one cell type. Muscle...

...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while...outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the experimental results. GeoCas9...could also be used with in vitro molecular bio applications that require high temperature reaction conditions...

...the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...it was first published in Nat Biotech in May 2016 (1), NgAgo has received much attention and the plasmid...Pooran's NgAgo Survey Survey results As of August 1, 2016, a total of 165 researchers responded to the...the cell, the same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's... dreams of a sunny day in Edinburgh.   References 1. Gao, Feng, et al. "DNA-guided genome editing using...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...survey. When asked if they could detect indels (an indication of gene disruption ability) with NgAgo, one respondent...

...premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa, NIH3T3, CHO, COS-1, 293HEK, Jurkat T cells Complexes...effective replication. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or...Concentation*** Blasticidin bsd  HeLa, NIH3T3, CHO, COS-1, 293HEK Inhibits termination step of translation ... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for... their bacterial counterparts, including how replication occurs and whether selection is necessary for...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are...

...them with TAA (7). This modification allowed the deletion of release factor 1, the protein responsible...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation...three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open’ codon ... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing... can follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...

...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton... and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https...

...transformation (Figure 1). As this approach requires no special reagents or purification kits, it is low cost...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...

...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class... than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ... this publication at Addgene. Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas...

... Table 1: Key differences between original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE...by PCR or next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. ...ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or ...or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs...The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...

...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...

...Subscribe to CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic ...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...use, functional in most species, and has many applications. That said, SpCas9 is not the only game in town... makes this molecule unique, and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving...

...directed base excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org/10.1038/s41467-021...predictable by machine learning methods. Nature Communications, 12(1), 4902. https://doi.org/10.1038/s41467-021...for more details on how they work!) Figure 1: Base transitions are edits between the purine bases...R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%... the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked (RBMX...transversions in human cells. Nature Biotechnology, 39(1), 41–46. https://doi.org/10.1038/s41587-020-0609-x...

...expression inducible by red light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline...Genome Engineering Visualization and Tagging Purification Controlled Expression Reporter Plasmids Reporter...Reporter Plasmids Tagging and Visualization Purification Controlled Expression Reporter Plasmids You may...Bacteria? E. coli are commonly used to store and replicate plasmids of all types, but beyond that, researchers...sites within this plasmid. Return to Top Protein Purification Sometimes looking at a protein inside cells ...and, epitope tags can be used to simplify the purification process. Using plasmids in this collection, ...those tags from the purified protein. Protein purification can be challenging, but once you get it right...

...animal, those B cells are fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called...antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting...cells and antibody-producing B cells that cannot replicate, meaning that only hybridomas will survive this... Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR Journal 46:... the Addgene blog Learn more about research applications for antibodies in Intro to Antibodies Read about...

...that have now become hallmarks of ideal FP timers: 1. Ratiometric determination of expression time The ...the FT series matures from blue to red (see Table 1 for emission and absorption spectra). Subach et al...times and varied blue-red maturation times (Table 1). These separate proteins should be useful for monitoring...cellular events occurring at varied time scales.  Table 1: Monomeric FP Timers and Associated Plasmids from ... know in the comments section below! References 1. Terskikh, Alexey, et al. ""Fluorescent timer": protein... (green or blue) within a population of timer indicates the age of the pool: the higher the ratio, the...-FT-N1 pTRE-Slow-FT pBAD/HisB-Slow-FT -  Applications of fluorescent protein timers FP timers have...

...evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...genome editing technology that allows precise modifications of DNA (replacements, small insertions, and ...configurations can be used to introduce specific target modifications. Since this means we could design hundreds to...

... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break...traditionally have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently...breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S....cut site location Position your CRISPR cut site as close as possible to the knock-in location. HDR depends...sequence to be incorporated at a precise genomic location, with little to no room for error. It’s no surprise...nutrients in their media, they will not undergo DNA replication, so make sure you are keeping an eye on those...

...transcription activation system co-opted from yeast (1). It is a Drosophila geneticist’s main workhorse to...he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression... A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire,...Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z. Marka...as overexpression of each gene individually. Applications of the Gal4/UAS system There are a number of...lines. In the meantime, be sure to browse the publication references and web links from my first post and...

...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit and -...dyes are also commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug...