...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration... points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for... the data points generated by replicate values provides useful information about the accuracy of the curve...If the unknown sample’s measurement is too low, redesign the standard curve dilution series with lower ...

...three-step approach for designing an experiment to identify the critical NANOG binding sites: 1. Find Enhancers...in combination with the techniques from Table 1, provides us with powerful GPS-like techniques to explore... chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...genomic coordinates into meaningful experiments. Table 1: An emerging family of "GPS for the genome" techniques...enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and set...histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties...

...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing oligos that avoid activating...activating MMR: 1) use a strain of bacteria that has key MMR proteins knocked out or 2) specially design ssDNA ...  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds- and ss-DNA Uses in Recombineering...Considerations dsDNA Insertion or deletion of >20 nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase... ssDNA Point mutations or changing a few nucleotides 0.1-1% or 25-50% when activation of MMR is avoided...from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ ...

...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific promoter or enhancer...

...from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from 1:1,000–1:100,000...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). You’ll want to...recommended dilution range is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations...bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure...Membranes look the same from both sides, so it’s quite easy to think lane 1 is‌ lane 8. Common ways to keep...reagent (usually an antibody).      Figure 1: A protein gradient. The density of the bands, which...

...protein design goals, both from the Baker Lab and others. The introduction of de novo designed proteins...protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize. Created with BioRender.com...concept that this kind of protein design is possible. Although Top7 was designed simply to be stable and novel...Hassabis and John Jumper! The Baker Lab focuses on designing entirely new proteins, improving our understanding...team was the first to develop and publish a human-designed protein that was not based on any existing protein...least that we’ve seen so far!). They started by designing a novel topology, then iteratively generated 3D...got closer and closer to achieving it. Once they designed the protein computationally, the researchers expressed...

...) element and the cis-acting EBNA-1 from Epstein-Barr virus. EBNA-1 binds oriP and allows for replication... cell reprogramming factors and wait for cells to de-differentiate. However it’s difficult, especially...Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods for Generating ...-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...  Replicating oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These plasmids carry the origin...method Most iPSC research falls into two categories: 1) studies focused on better understanding the mechanisms...iPSCs experiences in the comments below! References  1. Sano, Masayuki, Minoru Iijima, Manami Ohtaka, and...

...interaction fades away the longer we are on it – so it is important to take breaks. After 1 hour (or another...those ideas and build from those.   Figure 1: Virtual lab meeting? No problem! You can still creatively...salmon_phd References Frassl MA, Hamilton DP, Denfeld BA, de Eyto E, Hampton SE, Keller PS, Sharma S, Lewis ASL...in scientific research and in any workspace. Re-design lab meetings with a focus on discussion and brainstorming...pivotal in writing publications/grants too. As described by several works (Wuchty et al., 2007, Vicens ...another designated time) of meeting, someone could raise their hands or signal the approach of a brain...prepares a coffee, or simply moves away from the desk to switch off a few minutes. The host could also...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...effector was developed, called Cas7-11. Despite being in Class 1, Cas7-11 is a single protein effector ...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-...

...pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive control necessary...because the loading control band is weaker in condition 1 than condition 2, the normalized values for the two...to using loading controls accurately. Assumption #1: The loading control is not perturbed by the experiment...without your knowledge? If you can’t trust Assumption #1, any change in the normalized intensity of your POI...interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears... the protein abundance axis, both blue “condition 1” points (POI and loading control) are smaller than...assumptions discussed above into question. Assumption #1: The loading control is not perturbed by the experiment...

...was around 33% of that observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation...paper. Green and red lines depict the sum of mean +1 and -1 std for each paper, respectively. The fitted...complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where there were zero plasmid... from 5/22/14 to 5/19/20). Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict the ...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...are no exception to this effect -- meticulously designed experiments in labs around the world were put ...

...loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion into loop 1 caused...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...favorite protein (YFP). You have two options:  Option #1: Affinity tag purification You tag YFP and use an ...and one against GFP. Insertion of AsLOV2 into loop 1 always generated OptoNBs with dark-induced binding... induced dark-induced blue-light induced Table 1: Summary of the effect of AsLOV2 insertion site on...purification is now a reality thanks to OptoNBs. Recently designed by the Toettcher Lab, OptoNBs are photoswitchable... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...

...assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central PMCID: PMC2822740...with minimal spectral overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein... enabled labeling of particular structures (Table 1). Each construct was flanked with a CMV promoter (...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the ability ... all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular Structures ...maxima Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2...

...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ... strains and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid ... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME approach. Figure from...genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2...LaFleur, et al. 2024.   The X-CHIME deposit also includes pXPR_071, a plasmid that leads to higher titer...blog CRISPR101: Validating Your Genome Edit How to Design Your gRNA for CRISPR Genome Editing CRISPR Cheat...

...7.1 (2014): 1–7. PubMed PMID: 26224555. PubMed Central PMCID: PMC4884002. 10. Boer, E. de, Rodriguez, ...attach proteins or polypeptides to a solid substrate (Holden and Cremer, 2003). References 1. Chapman-Smith... of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg yolk while its binding ...matrices.” Journal of Nanobiotechnology 13.1 (2015): 1–14. PubMed PMID: 25980573. PubMed Central PMCID: PMC4461904...Northern blotting Cell surface labelling Attaching polypeptides to a substrate Flow cytometry/fluorescence-activated...your downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications...

... mutagenic primers to introduce all desired codon changes (step 1 in the image above). A second universal...Supplementary Note 1 in Wrenbeck et al for details on how nicking was optimized. How should I design my mutagenic...making some or all of the following modifications: 1) increased concentration of Taq polymerase, 2) increased...introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil labeled wild-...strand is nicked by the BbvCI variant not used in step 1. Then this strand is degraded with ExoIII and ExoI... the first digest (step 1 in above diagram). Remember that the BbvCI enzymes nick the DNA strand that ...time no, but there are 2 instances where it does. 1) When the backbone vector has multiple BbvCI sites...

...indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid 12259: pMD2.G (depicted on...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...Strausbaugh LD, Gray P, Vartak NB, Brown M, Talbot D, Berg DE. pBR322-derived multicopy plasmids harboring large...Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761-340-4_2. PMID: 19763938. Maucksch C, Bohla A...confirmations like linear and nicked plasmids, as described in this article.   Long-read sequencing Addgene...

...vectors without specific facilities (Biosafety Level-1). Other possible tools, lentiviruses for example, ...suspected cases of insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant...Subscribe to Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...the right serotype? Keep asking questions!  Table 1: AAV Serotypes and Tropism Experiment  Detection...the vicinity of the injection. Examining serotypes 1, 8 and 9, not only do they undergo retrograde and ...types of research may be enabled by the use of AAVs? 1) Combined functional and anatomical mapping of circuits...

...workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated CD365 in the most...dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1%...antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when...to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a...cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus...between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize...

...mediating the entry of SARS-CoV and SARS-CoV-2 viruses. ( 1 ) TMPRSS2 - a serine protease that primes the SARS-CoV...SARS-CoV-2 Viral Pseudotyping Collection - This page includes a table of all spike expression plasmids in our... industry is continually growing. ID Plasmid Description Industry PI Return to top Other Coronavirus Plasmids... SARS-CoV, MERS, BAT-CoV Plasmids ID Plasmid Description Industry PI Due to export control regulations...plasmids related to these genes below. ID Plasmid Description Industry PI For more information on these genes...CRISPR-Cas13: Open-access SHERLOCK research protocols and design resources (Link opens in a new window) Find Zhang...COVID-19 research in the table below: ID Plasmid Description Industry PI Do you have a suggestion for plasmids...