...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...were derived from the same source and were incubated on the same plate at the same time. Although the replicate...separate wells (within the same plate) with purified plasmid from the same aliquot/preparation. The replicate...provide the baseline/standard against which other samples can be compared. The Empty Vector Control (mentioned...control in this experiment could be an shRNA—in the same backbone as Plasmid A—that does not target any mammalian...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials... the ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate while expressing.... While these two strains ultimately perform the same function, there is some evidence that ccdB Survival...

...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations...used method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...Statistically defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. ... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...individual values match across the board. If the samples or sample conditions change, the multiplexed protocol...validation data, be aware that different machines, samples, and treatments may affect the assay. It’s unusual...

...from the same species and 2) these fragments will be more likely to bind (and block) the same epitopes...epitopes recognized by the secondary.   Figure 2: There are a few common solutions to avoid background...only antibody option is from the same species as your tissue samples. The bad news: this situation is ...excess secondary with serum from the same species as your primary/sample, and then applying this mixture to...processing and sample preparation can also help reduce the levels of endogenous IgGs in your samples, but know...The culprits are endogenous IgGs in your tissue sample. Most common IHC protocols use two antibodies -... the first antibody. When endogenous IgGs of the same species as your primary antibody are present, your...

...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...NLuc requires 100-fold less protein to produce the same amount of light from an Fluc or RLuc reaction. In...protein that could be co-imaged with GFP using the same wavelength for excitation. The result was the cyan-excitable... K., et al. (5) imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs...

...counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview of an...into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques, ...recognize the species of your primary antibody and 2) is conjugated to a fluorophore that suits your experiment...multiple proteins relate to one another in the same sample. For this strategy to work, it is important to...you use will depend on the target and sample type. Sample Preparation This step can involve multiple...target and sample type. Primary Antibody This is the first antibody applied to your sample, which will...of cells found in my tissue.” It is based on the same principles as other antibody-based assays, like ...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative...application Collect and lyse samples to make proteins available. Tissue samples may need additional processing...culture samples to extract proteins. Homogenize (tissues only) and lyse. Cross-link the samples with formaldehyde...

...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially... roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...possible, use low-bind or siliconized tubes to store samples as it is more difficult for viral particles to ...

...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes for in...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...

...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first...contact Addgene. We may require that you send your samples to Addgene pre-transformed in this strain. ...with instructions on how to prepare and ship the samples to Addgene and contact your technology transfer...

...to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...of plasmid into a separate daughter cells (Figure 2) (Ebersbach et al., 2005). However, more recent studies...Plasmids. Microbiology and Molecular Biology Reviews 62(2): 434–464. Pubmed PMID: 9618448 Diaz R, Rech J, Bouet... Plasmids with the same replicon are incompatible because they compete for the same replication control...incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding...if they have the same centromere-like region, and the NTPases end up “walking” the same plasmids to one... when multiple plasmids within one cell have the same replicon and/or partitioning system. Let’s start...

...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...

...fluorogen activating protein - dL5** in this case), and 2) a fluorogen (the TAP - targeted and activated photosensitizer...cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...PMID: 26808669. PubMed Central PMCID: PMC4916159. 2. Jacobson, K., Rajfur, Z., Vitriol, E., & Hahn, K....exposure times (5 minutes vs 10 seconds) to achieve the same level of killing as Mg-2I-dL5**. Mg-2I-dL5** also...

...released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce the same unique SapI 5’ overhang...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...

... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...at the same location that their host colonies grew. If your cells of interest stick to the same location...

...lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous indel ...Denham lab made several other small changes creating 2 other versions but these did not surpass GEmCherry2...fluorescence and in-frame fluorescence.  Figure 2: The GEmCherry reporter. Image from Højland Knudsen...resistance gene followed by the target site (the same sequence you’d like to mutate in the genome) and...

... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to assemble the  fragments...

...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure in ...ReFigure by creating ReFigures and answering this 2 minute survey! You can also find ReFigure on Twitter...another paper. A large fraction of readers cover the same ground again and again as they don’t save the insights...disease or therapeutic Side-by-side comparison of the same experiment from different cell lines/model systems...

...groups (4; brighter than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice...future.  Many thanks to our guest blogger Samantha Young. Samantha Young is a Medical Writer with a PhD in... This post was contributed by guest blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has...technique, you can assess several sgRNA’s at the same time, and choose the most effective ones to increase...