...across the board. If the samples or sample conditions change, the multiplexed protocol may have to be ... If there are multiple amplicons, or primer dimers forming, the curve will show multiple peaks.    ...optimizing for LightCycler machines.) Multiplexing It is possible to multiplex qPCR when using fluorescent probe-based...individually run sample when developing the protocol and optimized until the multiplexed and individual ... With the right conditions and parameters, some people find it can detect even smaller differences, and... can be the more optimal choices at times. For example, chemistries that rely on hairpin design of probes...the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison...

... worked with people outside the department to accomplish a variety of goals. For example, I worked with...experts that complement your (scientific) skill set and is therefore very useful for complex projects. It...met interesting people at conferences and you have successfully reached out to people outside your organization...Meeting people outside your organization just to have a quick chat can be rather tricky (think people based...Addgene I work with people from various organizations in science and beyond. For example, I help organize...collaborations are becoming increasingly important as more complex approaches to scientific questions become popular...mutual benefit.  Our partnership with SnapGene, for example, allows us to display high-quality maps and annotations...

... 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...

...cells or tissues contain a complex mix of proteins seen as green squares, purple circles and blue triangles... adjusted depending on the sample. It is important to remember that sample lysates will also contain proteases...capture antibody. This will deplete the nonspecific proteins from the sample prior to beginning the IP....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 ...

...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...channel to another. When you label your samples with multiple antibodies, the fluorescent probes on the...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...overlap in spectra is often called "spillover." For example, if your flow cytometer reads out the fluorophores...

...your samples. There are a few issues that can arise in this step: protein degradation, incomplete lysing...time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...wells. Most people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill ...proteins going completely through the membrane or larger proteins not transferring completely onto the membrane...your protein gradient, compare your diluted samples. If multiple dilutions are showing the same signal (Figure...amount of protein/sample loaded Incubate primary antibody at 4 °C overnight Incomplete washing Ensuring...gradients. Protein gradients use dilutions of your sample or protein of interest to understand the dynamic...

...we just made the sample bigger? In ExM, and its various derivatives, biological samples are enmeshed in...label) your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link...times, and the physical properties of the sample itself. For example, cultured cells are typically easier ...structure. For example, cytoskeletal structures that form continuous lines in pre-expanded samples can sometimes... popularity with an abundance of protocols and examples of applications from across disciplines now available...the number of specific targets you can label in a sample. Super-resolution microscopy (SRM), a family of... its original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This expansion...

... permeabilize a sample. But you can also use organic solvents after fixing your sample with a cross-linker...during each step. Now that you know the basic principles of IF, we’ll take a slightly deeper dive into...protocols. Here we’ll start with just the first couple of steps - mainly, fixing and permeabilizing. ...the point?  Fixation allows you to freeze your samples in a given state or point in time. You want to ...processes while maintaining the structure of your samples, the relationships between cellular components,... molecules and it is great for preserving your samples and maintaining cellular and tissue morphology....cross-linker for IF. However, you might come across a couple other terms that can cause some confusion. Paraformaldehyde...

...connections This is a site for meeting people. Use the settings to let people see who you are when you look at...has spam profiles and trolls. People don’t like to connect with complete strangers as it borders on my... a LinkedIn profile is absolutely necessary. If people can’t find you on LinkedIn, you will lose opportunities.... When I am speaking at a new institution, for example, I will often see a whole bunch of notifications... (unless they’ve chosen to hide it). While most people use LinkedIn messaging, not everyone likes to. ...opportunity to start things out right. Also, many people on LinkedIn don’t appreciate random requests without...results in a generic invitation. 3) Connect with people you have really talked to The best time is within...

...I’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot...each individual sample, this will reduce convenience and comparability because each sample with be quantified... low-concentration samples can also behave differently in PCR, even if both samples fall within the linear...reference sample of known titer, which is at a higher titer than the AAV RSM. By using these samples in all... titer values of new AAV samples. We also typically quantify our AAV sample by two qPCR assays and compare... viral vector preparation protocol, and we are pleased to report that over 95% of viral capsids in representative...Systems, Harvard University.    Which titer do people usually report? Addgene and other viral vector ...

...irivbrro ddPCR  Droplet digital PCR (ddPCR) works by using oil droplets to partition a single sample into thousands... reference sample with two copies of the gene (Ma, 2014.) As with all qPCR assays, samples, standard curves...see image, left, of droplet immersion technique, courtesy of CAR15.) Each droplet contains a primer and...reference sequence present in the same amount in all samples (usually to a ubiquitously expressed, one-copy ...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...

...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 ...

...size and density present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent...thiols, to cleave disulfide bonds. Load the samples, one sample per lane, onto the top of a resolving gel...simply the western blot, or western, is one of the simplest methods to detect the presence or absence of a...Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated in a gel, transferred...to detect a single, specific protein within the complex mix derived from cells or tissues. Westerns are...is the first step of a western. To prepare the samples for SDS-PAGE, measure the protein content and normalize...normalize to ensure equivalent loading. Denature the samples to their primary amino acid sequence by boiling...

...data from multiple primer pairs, one can infer the predominant state of registers within a sample using a...alternative. The qPCR method can be performed on a sample of multiple registers. However, it is only semi-quantitative...states in a sample; instead, it will suggest the predominant state of registers in a sample based on a ...methods do not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids... often times there are multiple register designs that may be used to implement the same gene regulation...needs. For more context, exposition, and detail, please refer to the paper. Why might one be interested... achieved by expression of other recombinases. Multiple orthogonal attB-attP pairs per recombinase may...

...sequence this highly complex hot spring sample. The sample was separated into smaller samples of 5-10 cells ... Mork collecting samples at the hot spring. Hiking all day and processing samples by night was fun...leave your samples behind! Paul and Anastasia (Potanina) Neddersen characterized a few samples by 16S ...from these extreme environments. Gearing up for sample collection at the hot springs My husband, Paul ... and two deep cycle batteries for power so the samples could be kept frozen after collection. Figure...challenges: collecting and processing environmental samples Since the high temperatures and unstable ground...dangerous, we used telescoping poles to collect water samples from a distance. At each collection site, we took...

...pipette samples in and out of it, you can set up a procedure for it using the Biomek FxP. For example, we ... pretty simple. By clicking a few buttons you can quickly tell the Biomek FxP how much sample to pick ...easy to spread out your samples. This can be useful if you’re transferring samples from 96 wells plates ... from many bacterial samples at the same time as opposed to preparing each sample individually. Enter ...provide you with some tips on how to automate some simple processes in your own lab. Automating bacterial...warming up too much and dying.  Also, we added a couple of platforms that fit 48 well q- trays allowing...mobile pods - one pod can pick up 96 tips for easy sample transfer between 96 well plates. The second pod...

...high-quality isolated DNA samples of sufficient quantity for sequencing. This process is completed in a plate format...MiSeq that takes about two days to complete. After the run completes, we begin our assembly process. Again...holes are then filled with other samples in our queue such as viral samples for Viral Genome Sequencing (... libraries.  Viral samples are filled into the same process as our normal Complete Plasmid Sequencing ...unique barcodes per sample so we can assign sequencing reads to the correct sample. The PCR is then performed...yielding anywhere from two to six plates of 96 samples per week. This is where our new process will begin...begin. Partnering with seqWell, we are using the plexWell technology to easily and quickly create Illumina...

...the primer sequences. Multiple plasmids can be included in a single PCR, for example for subcloning genes...perform multiple recombination events, all linear fragments need to get into the same cell. For simple cloning... deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type of...Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications... the multiply modified product. For example, by combining multiple primers, a C-terminal epitope tag can...GFP-tag (Figure 3). Achieving multiple modifications requires multiple recombination events to assemble...allow any cloning procedure to be performed using a simple two-step, 2 hr protocol prior to transformation...

...approach is as follows: Prepare the sample. Incubate the sample with the primary (unconjugated) antibody... but it probably should be. In a multiplex assay - one with multiple proteins of interest - the secondary... simplifying multiplex assays.  A brief note Since the indirect method relies on multiple secondary antibodies...antibody instead of the primary antibody. Since multiple secondary antibodies can bind to a single primary...in blue; secondary antibodies are represented in purple; signaling molecules are represented in light green...stem of an antibody’s Y) is large enough to allow multiple secondary antibodies to bind to a primary antibody...remove any unbound or excess antibody. Incubate the sample again with a conjugated secondary antibody, which...

... as Cascade (CRISPR-associated complex for antiviral defense) complexes, while Class 2 use single protein...tools using a Cascade complex is tricky, as you need to ensure every unit of the complex expresses at similar...present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with a...primary effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous... it can process multiple gRNAs under a single promoter, allowing for easy multiplexing. The double-stranded... of CRISPR technologies. Learning the general principles of this classification system will help you gain... your own research! Classification Structure Principles of CRISPR classification While CRISPR systems...