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...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...important is the transformation efficiency of the strain: when asking the cell to perform multiple recombination...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...adopted by any laboratory. All procedures are performed using the same three step protocol:  Fragment... encode homology arms to allow immediate co-transformation with linearized vector backbone. DpnI digestion...competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications will require ultracompetent...

...’s time to think big and perform your assemblies in a high-throughput format. If your lab owns a liquid... after each transformation and/or use different E. coli strains with higher transformation efficiency....in silico cloning tools When available, always perform your MoClo assemblies in silico before you start...original Addgene kit plate for fresh bacteria. Performing your Assemblies Calculate your volumes As you...Save time, go high throughput You are ready to perform your assemblies, but you have a high number to ...restriction–ligation reactions in a 96-well (or higher!) format. Liquid handling robots are not only faster and...lowers experimental costs. If you are planning to perform a high number of assemblies, you will also likely...

...bacterial transformation One protocol that has been automated is high throughput transformation. With our...a couple of platforms that fit 48 well q- trays allowing us to do up to 96 transformations in one run ...easier and more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar...deposits (over 100 deposits per month!), manual transformations were taking a lot of time. Enter the Biomek... We wanted a machine that would improve our transformation protocol and, after looking at a few options...run compared to doing about 50 transformations in the same amount of time by hand! Setting up a basic ... fine piece of machinery we have been able to transform 96 samples in about 4 hours with a greater than...

...anyone who has ever worked to sequence DNA on NGS platforms will tell you, while the process has become much...sequencing. This process is completed in a plate format yielding anywhere from two to six plates of 96 ...donated MiSeq.  For Complete Plasmid Sequencing we perform a 2x251 run on our MiSeq that takes about two days...involves a significant amount of QC. First, we perform Picogreen quantification on each and every isolated...samples for Viral Genome Sequencing (VGS). QC is performed throughout the library preparation process and...Aligning the sequence The first step we take when performing quality control of newly deposited plasmids is...detecting common features using Snapgene. If we find information that differs from what we would expect given ...

...purification before transformation. After transforming cells, you should perform colony PCR with vector..., ligation, and transformation. While restriction cloning is still routinely performed by many labs, a...products are then transformed directly into chemically competent E. coli and transformants selected with ...+ + Gel Purification + - Ligation + - Transformation + + The FastCloning process in brief In ...for this procedure to work properly. Directly transform chemically competent cells with the digested mixture...-cycle PCR leads to the most colonies after transformation. When a 1:1 vector:insert ratio is used in ... and 2 µL of digest product are used in the transformation, >70% of the colonies are correct. Good luck...

...AMA   Guidelines for giving feedback Use formal and informal mechanisms for feedback (more on this below...her management style. Formalizing the feedback session Giving regular, informal feedback is a very effective...Using this standard format not only enables you to discuss a person's performance or what has been learned...scientists seem to find it easier to give feedback in formal sessions using a specific list of questions after... it helps to plan effective next steps. For a formalized feedback session, answer the questions below ...discuss openly. Both parties get used to using this format and it becomes even more effective over time if...of indirect language You must be honest about performance problems. While giving corrective feedback isn...

...organizational performance and often have to be able to quickly synthesize information from a variety ... their visual format is quite appealing to many audiences. While all of these platforms were developing...not hosted on the platform, they can be hosted on YouTube, Vimeo, and other platforms (such as university...quickly. Taking what I loved from each of these platforms (such as social networking, visual appeal, instant... across multiple other sites), I created a new platform for sharing video abstracts about research: www.WeShareScience.org...www.WeShareScience.org. WeShareScience is a free platform for sharing short, 3 to 5 minute videos about ...university websites). WeShareScience is a social platform that provides links to the videos, keeping the copyright...

...The ability of our users to discover all this information, however, did not improve at the same pace as...as the quantity and quality of the information. As a result, resources on the website were hard to find... than that: the general “findability” of the information on the site. Different users have different strategies...paths, the organization and completeness of our information, and the accuracy and relevance of our search...the advantage of a very specialized domain of information so we were able to fine tune our approach much...than if we were dealing with general purpose information. Below are a few of the strategies we used in...Categories We created a categorization of our information in order to more granularly customize the search...

...optimization as each antibody performs differently and some antibodies impact the performance of other antibodies...beautiful results. Performing an immunofluorescence experiment The process to perform IF can be broadly...important considerations to make when planning and performing an IF experiment. What is immunofluorescence... different species and colors makes it easy to perform multiplexed IF. By using multiple primary antibodies...in IF experiments. Precisely which controls you perform will depend on your experimental context.  Choosing... to use, then the primary antibody will likely perform as expected in both.  How do you find validated...may be, it is always important to verify their performance in your hands and in your specific experiment...

...80) are transformed to 100. This transformation (illustrated in the graph below) is performed because ...order reagents Perform your wet lab experiment Analyze the data For more detailed information on using CrispyCrunch...even more.) However, none of the tools we found performed batch guide design, batch primer design or batch... burden of having to manage all the standard information that goes into a 96-well plate and comes out ... reports. With CrispyCrunch, you get the all information from Crispresso plus a summary per batch. Ranking... at a genome by eye. We came up with a ranking formula that does just this: Rank = (compressed CFD score...the DNA target. CFD scores are then modified (“transformed”) such that weak interactions (e.g., scores <...

... base editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular...72 hours post-transformation/transfection in a transient assay. For stable transformation, regenerated ... reducing the likelihood of detrimental indel formation. After binding with DNA, the CBE deaminates a ...G)? You may wonder if a CRISPR-base editor can perform this change. However, there are no naturally known...deaminases (ecTadA), fused it with dCas9, and transformed it into E. coli harboring the defective CamR ...genome edited plants via Agrobacterium-mediated transformation. We also constructed smaller vectors suitable...suitable for transient assays using protoplast transformation. Cytosine base editors for plant There are several...

...to read a formulaic fiction story that doesn’t require any detailed retention of information or formulation... extraneous cognitive load refers to the way information or tasks are presented to a learner; and germane... particular way of organizing and connecting information and a logic to the types and timing of questions...question constantly (and so should you!)  Amount of information and level of detail Again, this is an obvious...load, than certainly amount of attention and information input. If you’re reading a book, you only have...quickly they can understand and process your information. A class of three year olds is unlikely to be...I would think about how I was presenting the information. Maybe I needed to remove a distraction, such...

...copies of your predicted sequence information. Collect plasmid information in a binder or digital notebook...barcoded tubes and plates. The critical pieces of information for a given plasmid are the backbone, inserted..., the predicted full plasmid sequence. Other information may also be critical to the maintenance and use... with us, it will be easy to find all of the information needed for the process. Always sequence verify... Once the physical sample and its associated information are at Addgene, we are ready to sequence verify...always be derived from a single colony to ensure uniformity of the culture. Sequencing directly from the ...concentration, and not all providers are equipped to perform Sanger sequencing from glycerol stocks, so make...

...confirmed or theoretical. No matter the level of information that is provided, each deposit goes through the...without the use of a reference sequence) using a bioinformatics pipeline developed at Addgene. The assembled...assembled plasmid sequence is then compared to the information provided by the depositor. If a depositor provides... will compare the NGS results to the plasmid information, other plasmids in the deposit, NCBI sequence... results and deposited sequences and plasmid information.  For each plasmid deposited at Addgene, QC scientists...scientist  The backbone elements based on the information provided and vector type  Any tags, selectable...sequence and assemble using the Illumina MiSeq platform, such as GC-rich regions and highly repetitive...

...feedback in multiple formats      Ask for input and opinions in meetings, via email, at formal communications... hand and eagerness to get, as well as give, information to make improvements. Members connect directly... this means encouraging a culture of maximum information flow so everyone knows what is going on and can...break, go exploring outside the team, and bring information back. No team has all the answers – reinventing...frequently One of the most important paths of information flow is the one that brings feedback back to ...from at least some employees. Watch out for "information allies" – these are employees in your group that...communications events or at informal gatherings, like at lunch. Many experts recommend using anonymous surveys with...

...pooled libraries have allowed scientists to easily perform genome-wide screens to effectively and efficiently...sgRNAs per gene. These libraries provide more information while using less resources and are particularly...that improved sgRNA activity and thus library performance. The Root and Doench lab’s optimized genome-wide...CRISPRi, and CRISPRa libraries have been shown to outperform other popular CRISPR libraries (see below). Let...colon cancer) cells and assessed the libraries performance via the depletion of a gold standard set of genes...developed sgRNA-level metrics to assess library performance in viability screens. Based on these metrics ...sgRNAs, Brunello even with only a single sgRNA outperformed another CRISPR library with 6 sgRNAS, highlighting...

...repository that can be used to perform screens in human cells. General information regarding how to use a CRISPR...assembly cloning platform called GMAP (Gibson assembly-based modular assembly platform). GMAP is based ...-wide Screening Using CRISPR/Cas9”. Detailed information regarding the Wu Lab library can be found in ...orphan GPCRs demonstrating the utility of this platform for drug discovery. Researchers can use plasmids...stop codon) and closed (including a stop codon) formats. The collection is compatible with both standard...Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of which greatly enhances the.... PubMed PMID: 26165496 GMAP - A new assembly platform for generating both simple and complex DNA constructs...

...containing full information about the suggested gRNAs to use, including all information provided in the...phenotypic readout, similar mapping could be rapidly performed by targeting all potential components of a signaling...available through Addgene. This means you could perform a screen with one library from Addgene, and then.... Thus, it is always a good idea to keep a bioinformatician close if you need help putting your list together... in vitro, but there is enormous potential in performing in vivo screens, studying genes affecting cellular...several algorithms. Please read the “Detailed Information” text linked to each reference library before...adapters. Output_UserInputParams: Contains all the information that you have input in the web interface, making...

...The QC team stays informed of emerging technologies in sequencing and bioinformatic analysis. At Addgene...-to-sequence plasmids, other sequencing and bioinformatic technologies are used.  Improvements to sequencing...sequencing technologies and bioinformatic analysis are occurring every year. At Addgene, we want to maintain...puzzle, the QC team compares it to the plasmid information or depositor-provided sequences to confirm the...the correct plasmid components. For more information on this process, see our blog post on NGS plasmid...cassettes of the same gene multiple times.  Bioinformatics at Addgene After a run on our Illumina MiSeq...workflow converts the raw sequencing data to FASTQ format, cleans and normalizes reads using the BBTools ...

...protocols and performing rigorous quality control (QC). The specific QC experiments performed varies for ...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...and stored at -80 °C. Titer Titering is either performed by Addgene or by the University of Pennsylvania...Pennsylvania Vector Core. In general, titering is performed by the facility that produced the viral vector lot....Pennsylvania Vector Core is (as of April 2016) also performed by ddPCR.  Quality Control Addgene ensures high.... To learn which specific QC experiments were performed on your lot, please contact us. Full sequencing...the Viral Genome Next-generation sequencing is performed on viral genomes isolated from the final AAV preparation...