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... 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...model, iterons of two separate plasmids bind to the same RepA molecules, forming a “handcuff” structure that...replication machinery. However, when plasmids have the same origin of replication, the negative regulation system...binding region compete for the same CBPs and NTPase to correctly partition plasmids to each daughter cell...

...compare different clones of the same target to see if the results are the same/similar, emphasizing the antibody...your sample to. For example, we use our negative control to compare exposure times of our samples. A prominent...confirming it will produce the same results even to a different sample. Finally, as a lab we discuss the...Baker, 2015), causing a huge waste of precious samples, time, and funds – up to US$350 million in 2015...noise. We then compare the negative control to a sample with the primary included to confirm that the signal...antibody and not from the background noise. If the sample images similarly to the negative control, that ...weak or with general non-specific binding. If the sample differs from the negative control, such as showing...

...holes are then filled with other samples in our queue such as viral samples for Viral Genome Sequencing (...preps and pooled libraries.  Viral samples are filled into the same process as our normal Complete Plasmid...unique barcodes per sample so we can assign sequencing reads to the correct sample. The PCR is then performed... process that yields high-quality isolated DNA samples of sufficient quantity for sequencing. This process...yielding anywhere from two to six plates of 96 samples per week. This is where our new process will begin...of a pipeline that takes the raw data from each sample in our pool and individually assembles the reads...quantification on each and every isolated plasmid sample and normalize each to fall within a 10 fold range...

...to a reference sequence present in the same amount in all samples (usually to a ubiquitously expressed,... reference sample with two copies of the gene (Ma, 2014.) As with all qPCR assays, samples, standard curves...housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set as the expression baseline...since you’re looking at the amount of DNA in your sample relative to your normal control - you can set up...number (RQ value) matches the copy number of your sample. In that case, an RQ value of one (or, more realistically... and the gene of interest to be amplified in the same reaction. Taqman’s increased sensitivity allows ...works by using oil droplets to partition a single sample into thousands of PCR reactions using Taqman chemistry...

...briefly boiling the sample before use or by directly adding a small volume of the sample to the PCR reaction...enzyme digest; however colony PCR can accomplish the same thing in less time and for less money. The key steps...also great for screening clones created with the same backbone but that contain different inserts. When...sequence of the insert is, allowing you to use the same primer pair to screen for the presence of many different...used to screen clones that were created with the same backbone because primers are plasmid dependent ...going to complete your colony PCR analysis in the same day, you can save the leftover bacteria-water suspension...experiment. The best controls for a colony PCR are the same ones used to verify if the colony PCR primers work...

... exact amount of protein in your sample, you’ll compare your samples to a standard curve. This curve is...the known samples is used to calculate the concentration of protein in the experimental samples. Get more...multiple proteins on the same cell simultaneously as well as compare between samples. Learn more about flow...of different isotypes can be used together in the same experiment because the reagents used to detect the... population of antibodies that all recognize the same epitope of the target protein. Most commonly these...expression in cell lysates, whole cells, or tissue samples. Many experiments that use antibodies use a primary...are used to detect the presence of proteins from samples containing a mixture of proteins. The proteins ...

...western blot requires a standard curve with a pure sample of your protein of interest (POI). This approach...divided by the intensity of the loading control in the same lane, and the resulting fraction is reported. ... band is stronger. The right gel has exactly the same POI bands as the left gel, but because the loading... a perfect world, protein concentration in your sample would directly correlate to protein abundance on....” By eye, the loading control appears about the same between the two conditions. But it isn’t! (No really...analogous green “condition 2” points by the exact same value. For the two solid POI points, this difference...detection range. As a result, they have almost the same value on the band intensity axis. This effect causes...

...and your sample(s), as you can only compare samples to a standard curve generated on the same blot. If...protein expression varies from sample to sample. To compare one sample to another, you’ll need to normalize...antibodies — mixtures of different rAbs that target the same protein — can be used to reproduce the benefits ...protein, and can require more antibody or loading sample, which may not be practical. Conjugated primary...Bradford assay or BCA assay. Be sure to aliquot your sample(s) before adding in your denaturing and/or loading... when you’re looking at proteins all roughly the same size, or a slightly more expensive gradient gel,...optimizing western blots. If you need to preserve sample, check the ingredients before using, as some contain...

... if you set up two identical assays with the same sample, you could end up with a titer of 4 x 1012 genome...standard curve is giving an accurate readout of your samples.  Of course, if qPCR were perfect there would be...must serially dilute your virus before adding your sample to a mastermix. At Addgene, we typically load 3... a wide range of titers. We usually dilute our samples 1:6 million to 1:25 million.  After making the ...and droplet oil that you use should come from the same manufacturer. Otherwise you will end up with poor...droplet quality. Using a droplet generator, the sample and the droplet generation oil are moved through...droplets is used to calculate the concentration of the sample. This concentration can then be used to calculate...

... into Sample6 as graduate students in Dr. Jim Collin’s Lab in 2009. From 2009 to today, Sample6 has changed...leadership, and grown to 25 members.  What is Sample6? Sample6 is an enrichment-free bacterial pathogen detection...current VP of Business Development & Operations of Sample6, for advice on how to take an idea developed during...etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages... the focus of the application was changed, but Sample6 still leverages the core engineering and scientific... embodied, and driving the enterprise value of Sample6.  “Concisely and effectively presenting the value...your audience you are doing amazing things."  As Sample6 raised more money, the team grew, and Mike stepped...

... and engineers were attending the same meetings and hearing the same words, they were leaving these meetings...aforementioned lab manager, said the word “sample,” did the word convey the same concept to Morgan, our senior engineer...help get everyone on the same page. Others in the meeting might also have the same question and asking can...When everyone at a meeting is using the same words with the same meanings consistently, meetings will be...counsel, used the word “service,” did he mean it the same way as Bryan, our customer support manager? When... an Addgene-specific definition. Words such as “sample,” “material,” “item,” and “format” all received...

...process at Addgene. All plasmids go through this same initial QC process using an Illumina MiSeq, but ...However, Sanger sequencing remains useful for small sample sizes and shorter regions due in part to its low...high accuracy. Conversely, you can only run one sample at a time, which can become costly and impractical...the bases (and, if necessary, separate different samples), the resulting data, or reads, are essentially...control. NGS is generally cheaper if you have many samples and can output more data, and it typically results...However, NGS might not be the best choice for small sample sizes, small regions of interest, or complex sequences... regions or for higher accuracy of a particular sample.  Long-read sequencing (“Third-generation sequencing...

...you only need 1-2 ul of sample and the run time is just a few minutes per sample. During the run, DNA fragments...absorbance. Then you measure the absorbance of the DNA sample.  These absorbance measures give you an idea of...especially when you expect low concentrations in your samples and are often used to quantify DNA for next generation...fluorescence-based methods require a standard curve, a set of samples with a known DNA quantity and their corresponding...That way, you can compare the fluorescence of your sample against this curve to quantify your DNA prep. Though...yourself as many fluorometers will calculate your sample concentration for you. Agarose gel electrophoresis...concentration of each band in the ladder. You then run samples of your DNA at different dilutions and quantify...

...105 signal intensity. If your samples and controls were processed the same way, the GFP- population should... control (left) or with the sample (right). Negative and positive samples are clearly distinguishable ...designed your first flow panel, you’ll load your samples into the cytometer and see one of two results for...Created with biorender.com. Pro tip! If your samples and control underwent different treatment processes...peak overlaps with the baseline suggesting this sample has a comparable expression to the baseline/negative...marker would be a good statistic to compare between samples. In cases where there is no clear distinction between...histogram plot and MFI values to characterize your samples may allow for better interpretation. Priyamvada...

...plasmid monomer from a plasmid sample, or if no monomers exist in your sample, you could try linearizing ... recombination between two or more copies of the same plasmid (Bedbrook & Ausubel, 1976). This recombination...restriction digest of multimers will produce the same fragment sizes and banding patterns on an agarose...Bedbrook & Ausubel, 1976). In fact, if your plasmid sample has a mix of different-sized multimers, you may...corresponding to each type of multimer present in your sample. In these instances, please note that the supercoiled...agarose gel). If the presence of a multimer in your sample remains unclear after visualizing it on a gel, ...perform whole plasmid sequencing of our plasmid samples during our quality control process. Since MiSeq...

...states in a sample; instead, it will suggest the predominant state of registers in a sample based on a ... use the same recognition site array on the register, and all three-input GRSMs use the same recognition...implement the same gene regulation program. We recommend testing multiple register designs for the same gene regulation...If an attB-attP pair is aligned (sites facing the same direction) on a register, then the result of their...amplify in some states but not others. Because samples of many registers will likely not be homogenous...infer the predominant state of registers within a sample using a metric that we describe in the Materials...alternative. The qPCR method can be performed on a sample of multiple registers. However, it is only semi-quantitative...

... that consistently receives around 200 new DNA samples every week. Here we will provide an inside look...own plasmids. Staying organized With hundreds of samples coming through our doors, the first step to ensuring...our deposit process, and then we carefully track samples through our lab using barcoded tubes and plates...process. Always sequence verify Once the physical sample and its associated information are at Addgene, ... will catch any errors made during the cloning, sample handling, or sequence assembly steps. All of our...This means that we are sequencing from the exact same stock that we store and distribute from. Of course...for the efficient handling of large numbers of samples. When we grow overnight cultures for glycerol ...

...membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing...you want to detect more than one protein in your sample? Or what if something weird happened during your..., almost like new. This approach preserves your sample and saves you from having to run a whole new gel...stripping solution (see below) to minimize loss of your sample from the membrane. Next, plan out the order of ... the weaker binding ones first to preserve your sample. Pro tip! Because some protein is lost each time...antibodies on it if you plan to reprobe! Just as your sample protein is retained better, antibodies will stick...even if round two is just another attempt at the same target!). In the stripping step, you’ll remove the...

...stained samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies...unmixing if your sample is stained with a PE/Cy7 antibody from a batch that is not the same as your single...fluorophore from another within a fully stained sample. Figure 2 is an example of the emission spectra...allows the user to use the autofluorescence in a sample as a fluorescent marker. Given that the level of... varies, it is critical to include an unstained sample for each tissue type you are running in your panel...control(s) Compensation or “reference” controls are samples stained with a single fluorophore. They’re required...antibodies are from the same batch, to avoid unmixing issues from batch-to-batch variation. Tandem dyes ...

...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...everything should be denaturing or annealing at the same temperatures. If there are multiple amplicons, or... here, here, or here).  If you’re working with samples that have not been documented in the literature...reaction should be compared to an individually run sample when developing the protocol and optimized until...setting it up for larger or frequently analyzed sample types and sets.   🔥Hot Tip🔥 The ABI machines...an RQ value representing the fold change of each sample compared to your control. But as we said previously...