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...CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems. The original pCas/pTargetF...dehydrogenase and generated a strain that could achieve glucose dehydrogenase expression that is 2.6 times... gene (cluster) interruption.     Plasmids for each of these systems are available from Addgene. If ...

...reactions are facilitated by the recombination of attachment sites from the phage (attP) and the bacteria ...coli strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The ccdB gene is present in the donor vectors ...toxic to the cell, Gateway cloning efficiency can reach >99%.   STEP 3: Express your Gene of Interest...

...techniques are used as part of a treatment plan, an approach known as multimodal therapy. Sadly, in many cases...the abdominal cavity. This targeted chemotherapy approach has been shown to be successful in killing many...as in the case of Paul Zygielbaum. A different approach to immunotherapy is bevacizumab (Avastin). This...

...specificity over a single SpCas9. The drawback of this approach is that two sgRNAs targeting opposite strands ...strands must be available in moderate proximity to each other.  If having two sgRNAs isn’t feasible for your...disrupt a catalytic residue. Good news! You can achieve your precise edit without even making a break.  ...

...the quantitative increase in fluorescent signal achieved when utilizing an optimized moxFP as opposed to...the Endoplasmic Reticulum and live cell imaging approaches. Erik is also an avid long distance runner, gardner...Griffith, her research focuses on the replication machinery of Kaposi’s sarcoma-associated herpesvirus.   ...

...the technique and ask if they would be willing to teach you. An essential, yet sometimes overlooked, aspect...transgenic rat technology is improving and may one day reach the sophistication of mouse transgenics. Everything...[10]. If used properly, optogenetics is sure to teach us much in all of these species. Happy experimenting...

...typically want to make sure your cells have first reached an appropriate density. Cells in the exponential...protein products, which can prevent the culture from reaching the desired density within a reasonable time-frame...cystosolic protein products, but this is not always achievable. Proteins which contain hydrophobic regions or...

...Blanking' measures the background inherent to the machine and your solvent. If using a NanoDrop to measure...A good purity ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy...

...HJs are resolved via cleavage in one of two ways - each junction resolution could happen on the crossing...Small edits require as little as 30-50 bases for each homology arm, but keep in mind these numbers may... PubMed. Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System. Li, K et al. PLoS...

...sciNote. Today, every lab has its own habits and approaches to record keeping. Top priorities in most cases...familiar paper-based lab notebook is still a standard approach to record-keeping in many labs. In a way, it will...digital data generated in labs. While many labs approach the situation differently, some adopting simple...

...based affinity purification and mass spectrometry approaches. Recently however, scientists have developed ...miniturbo Split-BioID: A conditional proteomics approach to monitor protein-protein interactions Split-..., et al. "Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally...

...Forward Design: We produced a video celebrating Massachusetts Nonprofit Day, which was the perfect opportunity...availability, a traditional solution was completely out of reach for Addgene.  So, onto research! I needed to design...mounted a monitor to it. It had an extension cord attached, with the option to mount a battery to it if necessary...

...Although there are different ways to ultimately achieve success, we find it is usually faster and easier...need to check for this in your system and then test each vector before starting your experiments (Rolland...A. (2021). Next Step in Gene Delivery: Modern Approaches and Further Perspectives of AAV Tropism Modification...

...University 29:31 - 49:45 - Reagent Sharing - Susanna Bachle, Addgene the nonprofit plasmid repository 49:45...future work cost both time and money with estimates reaching in the billions of dollars (Freedman et al 2015... not what most readers want to read. As Susanna Bachle points out in her talk (29:31 - 49:45), there are...

...efficiently into dividing immortalized cells lines but reach their limits when it comes to transferring DNA into...been established so far, with safety increasing in each generation. The 1st generation of lentiviral vectors...either tTA or rtTA (FUW-M2rtTA). In this kind of approach, a population of cells might express only the ...

...control? Addgene lists the primers used to obtain each result above the posted sequence in the "View Sequence...facility The corresponding primer sequence used for each reaction Tips and FAQs My sequence doesn’t match...

... strategies include using engineered ligases to attach a ligand to a peptide tag on your protein of interest...use the same tag for multiple proteins but label each separately before reconstituting, choosing your ...potential benefits and costs before investing in one approach or the other.   Finding the right tag for you ...

...the 3' end of the cut site until the first G is reached (shown in blue), at which point it will add back...transformation. Use 1-2 μl of annealing reaction for each transformation, following a standard protocol . ...

... SSB protein to improve accuracy and efficiency. Each enzyme has a specific and unique function for the...Complementary DNA fragments can subsequently anneal to each other. Phusion High-Fidelity DNA Polymerase - incorporates...

...cells into another cell type. However, these two approaches differ in several ways. Below is a table listing...after completing her postdoctoral training from Massachusetts General Hospital and Harvard Medical School....: 26042384. 27. Nejadnik, H., et al., Improved approach for chondrogenic differentiation of human induced...