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TypeBlog PostPublishedJuly 12, 2021, 1:15 p.m....Maya Kostman for the IGI. Here are some highlights of the plasmids shared over the last year and... more tools, head over to this blog post that highlights five different methods to use CRISPR to detect...
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Synthetic promoter AAVs for cell-type specific expression in retinal cells
TypeBlog PostPublishedMarch 12, 2020, 1:15 p.m....initiation sites of at least two genes with high expression and high cell-type specificity. Strategy #3 (ProC...promoter designs, promoters from strategy #4 had the highest rate of targeted expression. Surprisingly, <1% ...tracing experiments: While synthetic promoters with high rates of labeling are ideal of gene therapy applications... -
Cpf1 Update: Comparison to Cas9 and NgAgo
TypeBlog PostPublishedJuly 14, 2016, 2:30 p.m....NGG) in both distribution and GC-content, which might improve editing in AT-rich genomes. Additionally...editing efficiency for the Cpf1 orthologs was only slightly lower than that of the widely used SpCas9 and ...that AsCpf1’s off-target rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1... cleaves relatively far away from the PAM, NHEJ might retain the PAM site. Therefore, if HDR does not ... -
Plasmids 101: Terminators and PolyA signals
TypeBlog PostPublishedMarch 31, 2016, 2:30 p.m....elements. A high affinity terminator may be desired for multi-cistronic constructs where high termination...terminators, such as T7 and rrnB, as well as engineered high-efficiency terminators such as T0. Rho-independent...poly-uracil tract as shown in the figure to the right. The tertiary structure of the hairpin-DNA complex... -
Tips for Titering Your Lentiviral Preps
TypeBlog PostPublishedMarch 15, 2016, 2:30 p.m....RNA. Measuring p24 levels in a viral prep is straightforward and rapid as there are several commercially...Studies have found that values can be 10-1000 fold higher than for functional assays depending on the vector... collected lentiviral prep may be significantly higher than the same prep that has been stored in the ...routinely produce virus may want to choose more straightforward approaches such as the FACS-based or colony-forming... -
Degrading DNA with Cascade-Cas3
TypeBlog PostPublishedFeb. 11, 2025, 2:15 p.m....Cas effectors to light. One lesser-known Cas is waiting for its chance in the spotlight — Cas3. Cas3 can...Cas effectors can’t, while maintaining relatively high efficiency. These alterations are achieved through.... E., Bravo, J. P., Ramos, D., Hibshman, G. N., Wright, J. T., & Taylor, D. W. (2023). Structural snapshots... -
Plasmids 101: Degron Tags
TypeBlog PostPublishedMarch 14, 2024, 1:15 p.m....added to media, buffer, etc., which will bind and light up a tagged protein for imaging. Given its popularity... like the SMASh tag does. However, it is a straightforward system – HaloTag and its PROTAC are all that...tag Are all tags created equal? No! Choosing the ‘right’ tag does depend a lot on your unique experimental...function is easily perturbed by tags, then a small tag might be the way to go (dTAG/SMASh). If you don’t want... -
Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria
TypeBlog PostPublishedApril 4, 2019, 12:53 p.m....the Mobile CRISPRi cassette flanked by left and right Tn7 end sequences. Donor bacteria with a chromosomal...scheme. Mobile-CRISPRi integrates downstream of the highly conserved glutamine-fructose-6-phosphate aminotransferase...cell, the CRISPRi DNA (purple) flanked by left and right Tn7 end sequences (green) integrates downstream ...mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for genome-wide... -
Transferable Skills: Negotiation
TypeBlog PostPublishedMarch 3, 2022, 2 p.m....At Addgene, we've set out to fill this gap by highlighting that researchers learn many transferable skills...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...Although this approach to negotiation is the most straightforward, it can mean that everyone leaves disappointed... -
Starter guide to induced pluripotent stem cells (iPSCs) part 1: A renaissance in regenerative medicine
TypeBlog PostPublishedOct. 30, 2018, 1:04 p.m....of them: Characteristic ESC ASC Self-renewal High Low once isolated from the human body Usage Universal...specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC isolation. Low...post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient) For...each other in many aspects. Like ESCs, iPSCs have a high self-renewal rate and can be used universally to... -
Fluorescent Proteins 101: Fluorescent Protein Timers
TypeBlog PostPublishedMay 4, 2017, 2:30 p.m....organelle, and protein localization. For instance, you might dye your cells and look at them under a microscope...population of timer indicates the age of the pool: the higher the ratio, the older the pool. FP timers have ... al. Protein Excitation (nm) Emission (nm) Brightness pKa Maturation Plasmids Fast-FT 403 (blue),...sections are formed. In synthetic biology, FP timers might be able to tell researchers whether old or new cells... -
Google Forums Round Up: First Impressions of NgAgo
TypeBlog PostPublishedAug. 4, 2016, 2:30 p.m....but, to start, let's review why NgAgo is in the spotlight and take a moment to remember that NgAgo has only...a single experiment. Also, the NGG PAM sequence might not always be available at an optimal distance from...mean the technology is dead in the water. On the bright side, successful indel generation and epitope knockin...supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly degraded by the cellular machinery, leaving... -
Genome-wide Screening Using CRISPR
TypeBlog PostPublishedAug. 18, 2015, 2:30 p.m....being used to perform genome-wide screens and highlight some of the reagents that have been made publicly... library. Choosing the CRISPR library that is right for you There are several factors to consider when... Findings from this type of experiment can shed light on the mechanism by which cells gain resistance ... -
Plasmids 101: Multicistronic Vectors
TypeBlog PostPublishedSept. 9, 2014, 8:20 p.m....are studying a novel gene, is not always a straightforward process. Rather than try to directly detect...cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or.... (J Virol. 1988 Aug;62(8):2636-43.) PubMed. 3. Highly Efficient Multicistronic Lentiviral Vectorswith...):845-60. doi: 10.1089/hum.2008.188.) PubMed. 4. High cleavage efficiency of a 2A peptide derived from... -
Great Results Start with Great Standard Curves
TypeBlog PostPublishedAug. 15, 2024, 1:15 p.m....the unknown sample’s measurement is too high, start with a higher concentration of standard or make smaller...range is the linear span between the lowest and highest concentrations that the curve can accurately measure...labmates to get a sense of the concentrations you might expect to see for the unknowns in your experiment... -
CRISPR 101: Making a Knock-In Cell Line
TypeBlog PostPublishedDec. 22, 2022, 2:15 p.m....cell cycle rates; rapidly diving cells will have higher HR frequencies than quiescent cells. It is important...ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts are within... need to incorporate homologous sequence to the right and/or left of the knock-in site. The length of ...: 387-390 (2020). 10.1016/j.jbiotec.2019.11.010 Wright, D. W., Shah, S. S., Heyer, W. D. Homologous recombination... -
Tips for arabidopsis transformation
TypeBlog PostPublishedOct. 25, 2018, 1:23 p.m...., so it is pretty important to get this process right the first time. I’ll lay out some guidelines here...transformation process The process itself is straightforward. When your agrobacterial cultures are ready...s flowers inside. 50 mL falcon tubes or plastic weigh boats are both great options for this, depending... et al. (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs... -
Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus
TypeBlog PostPublishedMarch 17, 2016, 2:30 p.m....contributed by guest blogger Tessa Cressey. The highly pathogenic Ebola virus belongs to the group of ...necessity for working with this virus under the highest biosafety level conditions, BSL-4. In this regard..., it is important to choose cell lines that are highly transfectable. The amounts of each plasmid transfected...vary between labs, initial titration experiments might be helpful to achieve optimal results. It is important... -
Tips from the Repository Trenches: Using Barcodes to Track Samples
TypeBlog PostPublishedOct. 6, 2015, 2:30 p.m....samples that come through our doors. It would be a nightmare if we didn’t have a simple way to track all of....2%) transform the first time and can be stored right away. Every week, the lab performs an average of...bacterial glycerol stocks on Fridays. From each overnight culture, we prepare three sets of 2D barcoded ...for a way to organize samples within your lab, we highly recommend using barcodes! Barcoded tubes are easily... -
Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research
TypeBlog PostPublishedJuly 29, 2025, 1:15 p.m....CRISPR-associated nucleases are Cas9 and Cas12a, the heavyweights in the genome editing toolbox. However, both...particularly via viral vectors. While viral delivery is a highly effective method for gene editing and allows targeting... targeting mechanism. Small RNA elements called right-end RNA (reRNA) or omega RNA (ωRNA) guide the TnpB...structural scaffold derived from the transposon's right-end element, and a 20-nucleotide (nt) guide sequence...