... environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in plasmid...debris”; and then clean-up the plasmid DNA (Figure 2). For most kits, the steps for lysing cells and removing...together — no mixing and matching!   Figure 2: Plasmid isolation workflows are similar across kits... on plasmid preps, not all plasmid preps are the same. Your downstream application will dictate how much...plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if you get a ...you mix specially coated magnetic beads into your sample and use magnets to isolate the beads for the wash...situation as you can simply add more beads to your sample. But, you’ll need to ensure you have an appropriate...

...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...

...  AAV9, AAVrg pAAV-hSyn-DIO-mCherry 37825 AAV1, 2, 5, 8, 9 (20 µL size serotype testing) pAAV-CAG-GFP...size serotype testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP...size 20 µL samples of control AAV that scientists can use for serotype testing. These samples are available...serotype testing jGCaMP7c for calcium detection PSAM4 GlyR for chemogenetic inhibition Flp expression ...localization. The Sternson lab recently deposited PSAM AAV, which encode engineered ligand-gated ion channels...Plasmid Serotype Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase AAV Finally, we are... Plasmid Serotype Name 119472  AAV5  AV SYN PSAM4 GlyR IRES EGFP   Biosensors Plasmid Serotype...

... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for survival...between gene silencing and knockout methods of the same gene. However, recent work has called this prevalent...

...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A....human induced pluripotent stem cells from urine samples. Nat Protoc, 2012. 7(12): p. 2080-9. PubMed PMID...

...positive population. Figure 2: An FMO gating example. The FMO sample (A) can be used while setting ...otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would be difficult...It will be from the same host species, have the same Ig subclass, and be in the same fluorophore (e.g.,...controls An isotype sample is prepared very similarly to a FMO sample: you make a sample from a combined ...your samples. The single color controls should be used as a starting point, to preserve your sample, and...experiment. To make an FMO sample, you take a subset of your experimental samples, combine it into one well...controls will help you save on sample volume, so you can use most of your samples for flow analysis rather ...

...A-B+ cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive... the different populations. In the case of Figure 2, one population is B- and another two are B+. Our ...are in the lower right corner.      Figure 2: This flow plot has two populations that are B+ and... are represented as a series of dot plots in the same order. However, in an actual flow cytometry run,...parallel.  Let’s imagine we’re looking for cells in a sample that are negative for marker A and positive for... proxy for nucleic complexity of the cell.  The sample will have been labeled using a panel of markers... dots. In a contour plot, each line contains the same number of events. Thus, contour lines that are close...

...019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, R. J. (2022...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes used...vivo. The general process of these screens is the same: Deliver CRISPR library to cells of interest. Select...

...) 38 kb Cells HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources... a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https..., so the amount of cloning required is about the same between insect and mammalian systems. Baculovirus...P1 virus, so that if you ever need to remake the same virus, you can skip the first round of production...

...experiment using smFPs and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures...similar proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...super-resolution STORM imaging, and electron microscopy. Sample preparation for these techniques sometimes makes...smFPs retain their ability to be detected after sample preparation better than standard proteins tags ...

... is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with restriction...program) can then reproduce the DNA sequence (Figure 2). Accuracy and accessibility led to the widespread...biology98.3 (1975): 503-517. PubMed PMID: 1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new...searching for), then the probe will bind to the sample thereby labeling it. The label can then be visualized...strength of the signal in comparison to a control sample. Southern blotting applications range from confirming... MstII and southern blotting are conducted on a sample from an individual without sickle cell anemia, ... largely replaced by real-time PCR to answer the same experimental questions. Real-time PCR detects and...

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...general anesthesia (Taylor et al., 2016). These very same neurons also play a central role in motivated behaviors.... Hirsch, Matthew L., Sonya J. Wolf, and R. J. Samulski. "Delivering transgenic DNA exceeding the carrying...

... intensity of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W...SSC-A, so most people use that setting. Figure 2: As the cell passes through the laser beam and causes...scale in proportion (while H and W do not; see Figure 2), single cell events will show up in a straight line...When using flow cytometry to analyze your samples, it is necessary to set up a sequence of gates to be...the area under the resulting curve. By default, a sample on the flow cytometer (before you set the first...Practical example: lymphocytes in a mouse tumor sample You made it! So much about the theoretical background...gated out my lymphocyte population from a tumor sample. Many cell types can be found in the tumor microenvironment...