...in a PCR protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent...requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should...mutagenized plasmids. PCR primers (green) amplify the plasmid template (blue), and introduce a restriction ... flank the targeted region. Upon PCR amplification, the template is removed by DpnI restriction digestion...are designed to be complementary, but each primer may extend beyond the complementary region as long as...efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified fairly easily...extension time to match the size of your template. Amplification of GC-rich plasmids is facilitated by the addition...

...with each sample. Successfully analyzed samples will display the following parameters: Sample - The unique...multiplex sample, ICE includes visual representations of all detected edit types in the sample. The visualization...indel.   For a complete guide to ICE analysis, including detailed explanation and examples, read the paper...easy. Simply upload your Sanger sequencing files and provide basic information such as your sample names...the ICE software has two analysis formats: sample by sample analysis, which can compare up to five editing...results and a list of the analyzed samples (see below).   If the sample run had no issues, the analysis ...perform more in-depth analyses on each sample by clicking on the sample name or on its corresponding bar graph...

...generation simpler, as described in an earlier post on generating CRISPR mouse models. For simple knockout... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template. Although...ssDNA template and CRISPR machinery, Quadros et al. recommend ribonucleoprotein (RNP) complexes of Cas9...Easi-CRISPR. As in the knock-in example, you’ll design your repair template with 55-100 bp homology on each...plenty of Addgene plasmid options for templates - see the example at right using blue flame plasmid pmScarlet_C1.... It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical...

... the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety...-containing sample with their conjugated primary antibody. Users next incubate the sample with the multiwell...control, include a sample that is known to contain the target antigen. Some examples could be an endogenous...used to quantify the level of target antigen in a sample. While Engvall et al. originally developed the ... proteins and small molecules from a variety of sample types (Engvall 1971). Scientists value ELISAs for...). Fluorescent tags may also be used, but for simplicity we will discuss the more common enzyme-based ...capture antibody that binds to the antigen in a sample and then a second detection antibody binds. The...

...in liquid culture to amplify the library, followed by DNA isolation. The amplified library is verified ...your first step will be to amplify the library. This is similar to amplifying a plasmid: transform E. coli...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...impact what conditions it needs to properly amplify.  Amplification protocols therefore differ from library... for the libraries we amplify in house, but these are not general amplification protocols. Rather, each...your amplified library should be very similar to that of the original library. Like amplification, this...the size and complexity of pooled libraries, NGS is the best way to validate your amplified library and...

...you will need to start with a fixed sample. Depending on the sample type, this might be frozen or formalin-fixed...al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your sample, and targets, you... Figure 2: Examples of simultaneous multiplex IF labeling of rat brain samples from the James Trimmer..., or you can make your own. For example, to label the rat brain sample shown in Figure 2A above, Andrews... get completely washed away and leave no signal. If you see lots of extra signal in these samples where...set of samples where you label only one target in each. Compare these individually-labeled samples with ...referred to as immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC)...

...molecule in an unknown sample. To do this, you serially dilute a control sample of known concentration...extrapolate the concentration of an unknown sample. For example, a BCA assay uses a serial dilution of a ...concentration of the unknown sample based on its measured response. An example of this is shown in Figure...their bicinchoninic acid assay (BCA) to normalize samples before running a western blot. Standard curves ...control protein, incubated alongside an unknown sample with the BCA reagent. The absorbance of both is...you to determine the concentration of the unknown sample.  How do I make the standards for a standard curve...standard curve by serially diluting a known control sample, called a standard. The ideal standard curve has...

...detecting specific populations of cells in a complex tissue or sample (1). When combined with cell sorters, ...information about the cell’s complexity. A cell with a high degree of internal complexity such as extensive membranes...rule out dead cells and debris. For example, a whole blood sample will contain a mix of cells including...differences in their size and complexity, cells from a whole blood sample will separate into distinct populations...ensure that samples are single cell suspensions. For cells that tend to aggregate, pass the sample through...single-stained samples for each fluorochrome in the experiment. Single-stained samples must be as bright...experimental sample. In addition, you should include a panel of “fluorescence-minus one” controls, samples stained...

...protein:DNA complexes with antibodies specific to your protein(s) of interest. Once the complex is captured... Once you have your sample, the first step is to ensure your protein:DNA complexes will remain bound to...you’re working with a tissue sample, you’ll need to homogenize your sample before moving on to the next...the protein:DNA complexes. Once you’ve finished fragmenting the DNA, spin down the samples to remove cell...protein:DNA complexes and finally (5) isolated the DNA from your antibody:protein:DNA complexes. Your DNA...Homogenization for a tissue sample can also need significant optimization for the sample type and size.) It can...process is very similar for both, though tissue samples do require an extra homogenization step. For this...

...can test three or more groups (two samples and a control, for example) simultaneously, with up to two independent...differences exist between. For example, if an ANOVA analysis on a five-sample data set yields statistical...appropriate to use if all sample comparison is needed, without a high number of samples (~5). Tukey’s test ...test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal levels... variance among data.     Fig. 2: Examples of data distribution profiles.   Parametric...They are classic parametric tests and one of the simplest statistical tests available. Tailed t-tests If...you are solely interested in whether or not two samples are statistically different than one another, a...

...carrying sample-specific barcodes. Rather than continuing to process 100s to 1000s of samples individually...projects in many labs. However, the high costs of sample preparation for high-throughput next generation...sequencing instruments by enhancing the efficiency and simplicity of library prep. As part of our mission, we’ve...technology overview For any NGS experiment, the DNA sample being sequenced must be sheared into smaller fragments...workflows are limited by costly library prep, as each sample requires an individual library. The plexWell™ workflow...library prep”, in which a relatively large number of samples are subjected to a DNA-barcoding step that uniquely...labels DNA from each well of a 96-well plate. Then, samples from several plates are pooled together to create...

... adjusted depending on the sample. It is important to remember that sample lysates will also contain proteases... from the sample prior to beginning the IP.  During the IP, the target protein in the sample binds to ...proteins are completely removed. Wash steps should be performed even with a pre-cleared sample.  Eluting ...parallel with both a sample known to express the protein of interest, such as a sample transiently transfected...immunoglobulins, to isolate a specific protein out of a complex mix. Using this technique, users can look for the...the antibody or antibody-target protein immune complexes, wash away unbound protein, elute and measure ...Protein extracts from cells or tissues contain a complex mix of proteins seen as green squares, purple circles...

...the amplified region of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and ...extracted from a sample and subject to isothermal recombinase polymerase amplification (RPA) using a commercially... available amplification kit. In this case, regions of gene S and Orf1ab gene are amplified. RPA reactions...cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA binds to the complementary region in the...the samples. First, RNA is extracted and subject to reverse transcription. This is then amplified by PCR...cleavage. Highlights: Reverse-transcribed RNA samples are amplified by PCR instead of RPA. Unlike RPA, this ... RNA is extracted and subject to isothermal amplification by primers that target the E and the N2 gene...

... qPCR, viral DNA is amplified and monitored in real time. When the PCR is complete, the results are analyzed...required for a mini amplification reaction to take place.  When the PCR is complete, a droplet reader extracts...the fluorescence amplitude of each one. Droplets that fluoresce contain the amplified target sequence....cycles. After additional rounds of amplification, your fluorescence amplitude will increase, leading to greater...standard curve is giving an accurate readout of your samples.  Of course, if qPCR were perfect there would be...if you set up two identical assays with the same sample, you could end up with a titer of 4 x 1012 genome.... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...

... and dsDNA. This is not true for all amplicons; if your amplicon is AT-rich or has a secondary structure...individual values match across the board. If the samples or sample conditions change, the multiplexed protocol...Method Standard Curve?  Amplification Efficacy Calculation Amplification Efficacy Assumptions ... can be the more optimal choices at times. For example, chemistries that rely on hairpin design of probes...Allelic specificity Can be multiplexed Single amplicon validation technique Hydrolysis Probes (Taqman...Bustin, 2017 for a more in-depth review.)  Your amplicon should be between 70-200 bp long With ΔΔCT analysis...validation data, be aware that different machines, samples, and treatments may affect the assay. It’s unusual...

...need to amplify a library for your own experiments. Library amplification 101 Library amplification seems...successful amplification? To provide a point of reference, Addgene commonly prepares NGS samples using both...understood visually. As an example, the curves from our recent amplification of the Brunello library are...exciting and versatile offerings! We recently re-amplified our distribution stock of the Brunello Human gRNA...thought it would be a good time to talk about the amplification and verification processes we use to ensure ...taken to ensure that all of these plasmids are amplified and none are lost. Each Addgene library has its...its own depositor transformation and amplification protocol, but the overall workflow is similar for most...

...experimental samples. Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to...Figure 2: Compensation example based on experimental sample. Flow plots showing a sample before (A) and after...Compensation example with single color control. Flow plots showing an APC single-color control sample before... your experimental samples In order to do compensation on your experimental samples, you will first need...apply to your experimental samples as they did with your single control samples: if the population needs...fluorescent channel to another. When you label your samples with multiple antibodies, the fluorescent probes...overlap in spectra is often called "spillover." For example, if your flow cytometer reads out the fluorophores...

...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...adjust the settings with experimental samples. Figure 1: Example flow plots for a single color control...experiment. To make an FMO sample, you take a subset of your experimental samples, combine it into one well...population. Figure 2: An FMO gating example. The FMO sample (A) can be used while setting up your gating...  Figure 3: Isotype flow plot example. The isotype sample (a) can be used to confirm that your ...controls will help you save on sample volume, so you can use most of your samples for flow analysis rather ...

...you use will depend on the target and sample type. Sample Preparation This step can involve multiple...methods you use will depend on target and sample type. Example steps include: permeabilization, antigen...target and sample type. Primary Antibody This is the first antibody applied to your sample, which will...depend on the sample type, microscopy tools available, and your experimental goals. Example steps include...fluorescent molecules are applied to a cell or tissue sample where they bind to their targets, most commonly...antibodies to visualize a target within an intact sample, a cell in the case of ICC or a tissue in the case...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...

... can be completely denaturated (left) or complexes may dissociate  (right). B) Protein samples protected...protect fragile samples during cryo-EM grid plunge freezing By Rachel Leeson Cryo-EM samples can be damaged...and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered and evolved...antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment and structure determination By Mike ...for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki Lab...enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it easy...cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic...