... 79628 (C) Cv (capsid) N and C 79633 (N) and 79641 (C) NS1 (var: W98G) N and C 79635 NS3 C 79637...79637 NS2B C 79636 NS4A N 79640 NS4B C 79639 NS5 C 79632 (N) and 79631 (C) PrM N and C   Detecting...environment and relies on the fact that enhancers function independently of their position relative to a transcription...parts involved in altering gene expression are relatively well known, particularly in bacteria, the process...strains (5). MutL is highly conserved in distant relatives of E. coli so it can be used in a broad range ...The key to the biosensor lies in pH: DsRed is relatively pH-insensitive, while SEP fluoresces at cellular... and normalized to assess interactions. Higher relative firefly luciferase activity in the Co-IP compared...

...variation. Confirmation of sequence can be done from relatively cheap plasmid sequencing. While rAbs are monoclonal...to run a western blot. It allows you to detect relatively low levels of antigen, while providing flexibility...assay design. Conjugated secondary antibodies are relatively cheap and easy to source, making it easy to switch... not available in a pure form, you can perform relative quantification, which measures protein amounts...particular antibody, this truly does provide a relative, not absolute, readout. Normalization Overall ...purified protein not always available Calculate relative quantification Normalizing to ubiquitously expressed...length of your assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most...

...researchers also use bacteria like E. coli and their relatively well understood biology to answer many interesting...construct, grown at the permissive temperature (32°C) to allow transposition, and the construct is then... by growing at the non-permissive temperature (42°C). Transposition is efficient enough that selection...TALEN Gang Bao Gateway cloning and expression of a C-terminally His-tagged TALEN fused to an E. coli codon...protein fusion: contains empty vectors with N and C-terminal fluorescent protein tags that can be used... pET15-MHL 26092 T7-lacO (lactose/IPTG inducible) C-terminal TEV cleavable 6x His Cheryl Arrowsmith Empty...Lactose/IPTG inducible) Various combinations of N and C-terminal Strep and His tags Thorben Dammeyer Lactose...

...Biddle, M. S., Alende, C., Fotouhi, M., Jones, C., Ayoubi, R., Southern, K., Laflamme, C., Virk, H., NeuroSGC...T. M., Brown, C., Bandrowski, A., Virk, H., Edwards, A. M., McPherson, P., & Laflamme, C. (2023). Scaling....154034.1 Laflamme, C., McKeever, P. M., Kumar, R., Schwartz, J., Kolahdouzan, M., Chen, C. X., You, Z., Benaliouad...populations and to study relative protein expression. Its reliability will be entirely dependent on the selectivity...and the abundance of the epitope in the sample, relative to cross-reactive antigens – that is, an antibody..., H. S., Biddle, M. S., Smallwood, D. T., Weston, C. A., Castells, E., Bowman, V. W., McCarthy, J., Amrani...Biddle, M. S., Wright, A. K. A., Sousa, J., Weston, C. A., Chachi, L., Roach, K. M., & Bradding, P. (2019...

...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s. These...Compared to BE3, BE4 offers a 2.3 fold decrease in C→G and C→A products as well as a 2.3 fold decrease in indel... Hong, M., Zhang, D., Luan, C., Zhang, S., Wu, H., Gao, H., Bai, S., Li, C., Ding, R., Xue, N., Lei, Z... W., Levy, J. M., Yeh, W.-H., Zheng, C., Newby, G. A., Wilson, C., Bhaumik, M., Shubina-Oleinik, O., Holt...non-homologous end joining (NHEJ), HDR occurs at a relatively low frequency, especially in nondividing cells...) and adenine base editors (ABEs). CBEs mediate a C to T change (or a G to A change on the opposite strand...strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are base transition...

...You know qPCR is most commonly used to measure relative quantification of mRNA, which can be used as a...reported to work well in the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table... Accuracy is not as important as precision for relative quantification. A consistent 5% positive error...Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time...150 bp long Annealing temperature should be 60-65°C For qPCR, the important primer variable is the annealing... the primers.  Melting temperature should be ~60°C (between 50 and 65, with no more than 3 degrees between...curve will show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak...

..., Banh, L. M., Williams, C., Toh, M. S., Irby, M. J., Edwards, L. S., Lin, C., Owen, A. L. G., Künne, ... other Cas effectors can’t, while maintaining relatively high efficiency. These alterations are achieved...contains all five of these Cas proteins, while type I-C Cascades do not contain Cas6. Regardless of the subtype...Resources References Loeff, L., Brouns, S. J., & Joo, C. (2018). Repetitive DNA reeling by the Cascade-Cas3...Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5....Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., Donohoue, P. D., Rotstein, T., Kohrs,...inversions and large deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding...

...bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs...in size. The structure of the primer should be relatively simple and contain no internal secondary structure...pairs should have a Tm within 5°C of each other Primer pairs should not have complementary regions Note:...

...which had a total of 31 amino acid replacements relative to CFP484. Because the excitation and emission...combination results in a 2.6-fold increase in signal relative to mCerulean imaged with a standard CFP set. mTFP1...constructed 22 different mTFP1 fusions to both the C- and N- termini of the fluorescent protein and found... PubMed Central PMCID: PMC2292683. Shaner, Nathan C., et al. "A bright monomeric green fluorescent protein.... mTFP1 is also insensitive to physiologically relevant changes in pH. Find mTFP1 plasmids here! mWasabi...

...terminal); 2xNLS (C terminal) 47327 pET-28b-Cas9-His 6xHis (C terminal); NLS (C terminal) 62933...6xHis 6xHis (C terminal); NLS (C terminal) 62934 pET-NLS-Cas9-6xHis 6xHis (C terminal); NLS ...mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical...the stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR. Potential guide...

...Michael, S., Kuleshova, E., Ojala, D., Huang, C.-C., Gerfen, C. R., Schiller, J., Dudman, J. T., Hantman,...our in-stock viral prep service.  AAV9 AAV9 has relatively broad tissue tropism and high transduction efficiency...doi.org/10.1089/hum.2008.123 Dalkara, D., Byrne, L. C., Klimczak, R. R., Visel, M., Yin, L., Merigan, W....scitranslmed.3005708 Goertsen, D., Goeden, N., Flytzanis, N. C., & Gradinaru, V. (2022). Targeting the lung epithelium...Ishikawa, Y., Jae, L. T., Wosen, J. E., Nagamine, C. M., Chapman, M. S., & Carette, J. E. (2016). An essential... S., Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A...Horwitz, N., Beggs, A. H., Wagers, A. J., & Sabeti, P. C. (2021). Directed evolution of a family of AAV capsid...

...transversions C → TG → A A → GT → C C → G                   C → AG → C                   G → T A → C                   ...Rosser, S. J., Bi, C., & Zhang, X. (2021). Glycosylase base editors enable C-to-A and C-to-G base changes...deaminase could also perform cytidine (C) deamination, converting the C base to uracil (U). This is the same...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%...Beyond UNG: Optimizing C → G editors Now that several labs had shown that preferential C → G editing was possible...pyrimidines (C ↔ T).   Base transversion  Base changes from purine (A or G) to pyrimidine (C or T) or vice-versa...type of base editor invented. CGBE C → G Base Editor Converts C → G; also called a glycosylase base ...

...histidine amino acids on the N- or C- terminus of a protein. Neither the N or C terminus is preferable over ...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing...histidine residues expressed in frame on the N- or C-terminus of a recombinant protein of interest. The...need to decide whether to add the tag to the N- or C-terminus and how many histidine residues to include...or function of a single protein? Oftentimes, they rely on protein purification. In this strategy a single...contrast, the polyhistidine tag is very small and rarely affects protein function (Bornhorst 2000).  One...other affinity purification methods where binding relies on a specific protein conformation, the polyhistidine-metal...

...temperature of the PCR from 60° C to 61° C improved assay reliability. Different primers can have different...the ratio of genome-containing viral particles relative to the total number of viral capsids (which can...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...absolute titer values may not be as important as the relative titer values. Finally, because physical titer ... PubMed Central PMCID: PMC2957240. 5.Taylor, Sean C., Genevieve Laperriere, and Hugo Germain. "Droplet...that vary up to two-fold. How can we get more reliable AAV titering? For our viral service, we currently...vector preparations by qPCR. To ensure accurate and reliable results, we have optimized our assay in a few ...

.../000504294 Wang, T., Guan, C., Guo, J., Liu, B., Wu, Y., Xie, Z., Zhang, C., & Xing, X.-H. (2018). Pooled...rat, is another popular model organism. A close relative to the mouse, both are great mammalian models ...that make them an ideal model organism; they are relatively simple to maintain and breed and they have large...doi.org/10.13918/j.issn.2095-8137.2016.4.237 McCusker, C., & Gardiner, D. M. (2011). The Axolotl Model for ...elife.50651 Vieira, W. A., Wells, K. M., & McCusker, C. D. (2019). Advancements to the Axolotl Model for ...BSL-1 status, E. coli can be used as a model to relate these processes in more pathogenic bacteria (Blount...regeneration.  In addition, axolotls have many age-related phenotypic changes that are similar to those observed...

...for use in C. elegans. In 2017, the Korswagen lab developed Cas12a (Cpf1) for editing in C. elegans (Ebbing...editing from more sites of the C. elegans genome. Toolkits for editing in C. elegans The development of ...SapTrap-SEC kit for C. elegans genome engineering have also facilitated CRISPR editing in C. elegans. These...2018). To see more CRISPR tools for C. elegans, visit Addgene’s C. elegans CRISPR plasmids page. Future...Ruvkun for their seminal work on micro RNAs in C. elegans. The C. elegans work on small RNAs informed and drove... recovery of these edits. Initial work in C. elegans relied on selective markers such as drug resistance...possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms which I use to...

...bound to the C-terminal peptide. One of these C-terminal antibodies was Myc1-9E10 (aka anti-c-Myc [9E10]...sequences within the middle of the human c-Myc protein or the C-terminus, Evan, et al. isolated six different...that Myc1-9E10 and the other C-terminal antibodies only recognized the human c-Myc protein and did not recognize...experiments? Why Myc? When did the commonly used anti-c-Myc [9E10] antibody come into play? We’ll dive into...This protein had a highly specific antibody to the C-terminal five amino acids, meaning the tag could be...However, substance P has an amide group added to its C-terminus post-translationally, which turned out to.... As such, the peptide could only be added to the C-terminus of proteins of interest and proteins tagged...

...make your own. In my next post, I’ll explain the relatively straightforward process of generating your own...21356876. 5. J. Chow, Z. Marka, I. Bartos, S. Marka, J. C. Kagan, Environmental Stress Causes Lethal Neuro-Trauma...PubMed PMID: 28704652. 6. V. Rodríguez, D. Didiano, C. Desplan, Power tools for gene expression and clonal...Neurosci. 9, 703–709 (2006). PubMed PMID: 16582903. 8. C. J. Potter, B. Tasic, E. V Russler, L. Liang, L. Luo...

...37-16 °C cycling 4-base Bsal-HFv2 37-16 °C cycling 4-base BsmBI-v2 42-16 °C cycling... T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C 18 hours 3-base or ... T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C 18 hours 3-base or ...NEBridge® Ligase MM 37-16 °C cycling 4-base Bsal-HFv2® 37 °C Static 4-base Bsal-HFv2...Ligase MM 42-16 °C cycling 4-base BspQI, 1x NEBridge Ligase MM 42-16 °C cycling 3-base...Ligase Buffer 37-16 °C cycling 4-base PaqCl, 1x NEBridge Ligase MM 37-16 °C cycling 4-base...              37-16 °C cycling 3-base Sapl, 1x NEBridge Ligase MM 37-16 °C cycling 3-base ...

... 22nd International C. elegans meeting, I was again reminded of the extent that C. elegans researchers...and toolkits available at Addgene.  C. elegans open resources The C. elegans Neuronal Gene Expression Map...database specifically for C. elegans. This database provides detailed information about C. elegans 3’UTRs structures...The C. elegans community has always emphasized the need for open science and collaboration. The field... important for post-transcriptional regulation in C. elegans. To study the role of 3’UTRs, the Arizona...view the anatomy of worm layer by layer, the NeuroML C. elegans connectome, a model of the C.elegans neural...be used to simulate muscle tissue as it applies to C. elegans locomotion. To see the other resources included...