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We narrowed to 792 results for: LAT

Showing: 101 - 120 of 792 results

  1. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    Published
    Feb. 18, 2016, 3:42 p.m.
    ...colonies on the backbone alone plate and many colonies on the backbone + insert plate (or at least more colonies...backbone alone plate). Unsuccessful ligations will usually result in few colonies on both plates (unsuccessful...), in a vector alone plate with many more colonies than the vector + insert plate (unsuccessful 2), or... each plate (unsuccessful 3).     If you have a high number of colonies on your backbone plate (greater... Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification     ...throw the digestion mixtures together. You need to isolate your insert and backbone from the enzymes used ...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...

  2. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    Published
    Sept. 9, 2014, 8:20 p.m.
    ...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is...single-stranded RNA viruses to allow for the efficient translation of multiple genes from a single transcript. The...Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA. In the ...discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic cells.1,2 Since...they all have in common is the ability to spark translation initiation independent of the 5’ cap.  IRES elements...bicistronic expression; however, many could be easily manipulated to express more than two genes. Plasmid Name...

  3. What's New in CRISPR - March 2020

    Type
    Blog Post
    Published
    March 10, 2020, 1:15 p.m.
    ... via acetylation or demethylation. Casilio DNA Methylation Editing (Casilio-ME) is a methylation editing...human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene expression ...50–90% of average on-target DNA-editing levels relative to BE4max.  Read the paper in Nature Biotechnology...Michael Laub’s lab developed a CRISPRi system to downregulate expression of specific Caulobacter genes using...ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and the ctrA sgRNA... demonstrated the system can also be used to downregulate multiple genes at once. Read the paper in mBio...Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1...

  4. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    Published
    Oct. 10, 2023, 1 p.m.
    ... resistance genes so that you can specifically isolate bacteria containing your constructs of interest...bacteria as a way to produce more of the plasmid, isolate individual plasmids, or express a gene contained...goal of your experiment — to grow, express, or isolate plasmids. The presence of an antibiotic resistance...shaking at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase ...to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted...Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic... and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming ...

  5. Plasmids 101: Colony PCR

    Type
    Blog Post
    Published
    May 12, 2016, 2:30 p.m.
    ...highlighted below. Preparing template: Pick a single colony with a sterile flat toothpick or pipette tip ... clones for later culture: At this point, you will want to hang onto your clones for later use. There ..., just streak the colonies on an LB plate. You can use this plate to start liquid cultures. Lastly, you...polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze... to preparing a standard PCR reaction: combine template, primers, polymerase, and dNTPs and then incubate...released from the bacteria in order to serve as PCR template. Dealing with this and a few other colony PCR ...background colonies on your no ligation control plate. The more background, the more colonies you will...

  6. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    Published
    March 7, 2023, 2:15 p.m.
    ... vs. the factor that is being experimentally manipulated. Below are several core factors of statistics...t-tests Samples that come from the same subject or population - before and after a treatment, for example -.... Samples arising from different subjects or populations (a treatment group vs. a control group), on the...analysis, must be performed. These will be discussed later. One and two-way ANOVAs ANOVA tests can compare...Quantifying and controlling for any covariance between manipulated independent variables allows for a more accurate...how independent variables you are not directly manipulating affect an outcome (age, demographic, diet, etc...to know every pairwise comparison within your population? Sometimes this can’t be avoided, but if it can...

  7. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...furimazine. Right: Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates ...reversible, making PILATeS suitable for live imaging with a time resolution of minutes.  Find PILATeS plasmids ... F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization of monoclonal antibodies ...antigenic differences between human, canine and simian isolates. J Gen Virol., 68 (Pt 11), 2769–2780. https://... happen within a few hours. In order to use the latest mScarlet tags, mScarlet3 and mScarlet-I3, Oliver...for CRISPR/Cas9 in M. abscessus here! Neo D, Clatworthy A, and Hung, D. (2024). A dual-plasmid CRISPR...Bioluminescent reporting of mechanical forces with PILATeS By Emily P. Bentley Time to get flexible! Alex ...

  8. How to Be an Excellent Trainee

    Type
    Blog Post
    Published
    Sept. 26, 2023, 1:15 p.m.
    ... learned, and ask questions later – maybe even the next day. In the latter case, you can say “I usually...planning you can do to build a bench mentor-mentee relationship and bring your best self to the training experience...plenty of articles on building successful mentor relationships, asking for mentor support, and choosing your...the research group). The bench mentor-mentee relationship is extremely important – without a good set ...mentor than your scientific mentor. Thus, this relationship will probably define your experience in the ...Luckily, you can do a lot to contribute to the relationship dynamic and set yourself up for success! Getting...ll probably forget and end up asking your mentor later if you don’t write it down. And don’t be afraid ...

  9. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    Published
    Jan. 19, 2021, 2:15 p.m.
    ...chemiluminescence, precipitate the protein out of solution, or isolate cells expressing this protein. Read on to learn...antibodies against the injected protein. Several weeks later, blood from the animal is drawn and the antibodies... hand, monoclonal antibodies are a homogenous population of antibodies that all recognize the same epitope...dilution, which produces an immortalized, clonal population of cells that produce a single variant of antibody...antibodies, recombinant antibodies are a homogeneous population. However, unlike monoclonal antibodies, which.... Western blots are frequently used to compare relative levels of protein expression between cell types... present. The reaction takes place in a 96-well plate, where primary and secondary antibodies are used...

  10. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    Published
    Sept. 29, 2020, 1:15 p.m.
    ...biologist tool box” for its common use in plasmid manipulation and genetics research. Plasmids are ideal tools...studying genetic modification for their ease of manipulation and their ability to be replicated. Because ...processes naturally found in E. coli have been manipulated for use in molecular biology studies. For example...BSL-1 status, E. coli can be used as a model to relate these processes in more pathogenic bacteria (Blount...rat, is another popular model organism. A close relative to the mouse, both are great mammalian models ...limiting attributes of rats is their difficult to manipulate genome and limited genetic data. To bridge this...biological processes and signaling pathways that regulate regeneration and wound healing are conserved across...

  11. The time and cost required to make a plasmid

    Type
    Blog Post
    Published
    March 23, 2023, 1:15 p.m.
    ... overall inflation of USD has gone up 19.8% in the same time period. For ease of calculations*, let’s ...well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any of the ...Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid. Doench’s lab in the...long does it take in total? While much harder to calculate, in both our 2018 and 2023 poll, most people said...much does it cost to make a plasmid? Doench also calculated the cost of consumable reagents used to make ...2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene, the consumables...s split the difference and put scientific inflation at… roughly 34.9%, which means that the same plasmid...

  12. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    Published
    July 14, 2021, 1:15 p.m.
    ...immunogen, then isolating and purifying the antibodies produced from its serum several weeks later. Created ...as KLH or Freund’s adjuvant is injected, which stimulates the overall immune response. Once the initial... antibody response starts to wane about a month later, the animal is given additional booster immunizations...contains the antibodies - is further processed to isolate the antibodies. Most commonly, antibodies of the...the IgG isotype are used for research purposes. Isolating polyclonal antibodies from the serum There are...are a few different ways the antibodies can be isolated from the serum. Protein A/G purification Protein...different type of purification can be performed to isolate antibodies only recognizing your protein of interest...

  13. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    Published
    Nov. 24, 2015, 3:30 p.m.
    ...reporter lines for more sophisticated experimental manipulations, saving the time and money needed to develop...control gene expression, he could then selectively manipulate only GFP-labeled cells. Once Tang and Cepko found...but specific system. T-DDOGs also successfully regulated expression of channelrhodopsin-2, commonly used...for optogenetics experiments in specific cell populations. Listen to Our Podcast Interview with Connie ...-DOGs The T-DDOG results showed that GFP could regulate transcription in a cell-type specific manner, ...Cepko were eager to see if they could apply GFP regulation to other types of proteins. They started with...system may make optogenetics in specific cell populations even easier. Advantages and other possibilities...

  14. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    Published
    Sept. 6, 2016, 2:30 p.m.
    ...specific genes involved. Downregulation of tumor suppressors and upregulation of proto-oncogenes ­­­can...while simultaneously stimulating growth and division. The combined effect is unregulated proliferation. Another...cases arise and millions of people die from cancer-related complications each year. Despite these grim statistics...cells still need to successfully infiltrate the circulatory or lymph systems to spread, or metastasize. Many...Many cancerous cells will die trying, as the circulatory and lymph systems are not ideal environments ...Ras researchers, one of the most exciting and relatively new resources is the RAS Pathway Clone Collection...set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) that represents...

  15. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...like to introduce the edit. Finally, you must stipulate what sequence to introduce, which is done through... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...is recommended you check your cell lines for HR-related mutations before attempting knock-ins by HDR. ...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...

  16. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...enzyme specifically cuts methylated DNA, so it will selectively destroy the template, but not newly synthesized...-55 °C for the homologous region (calculated using Oligo Calculator). Insertions - To insert short sequences...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive colonies...for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25 μl single-tube...Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while modifications...sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature (Tm... bp, such as epitope tags, design primers with template binding regions to amplify away from the insertion...

  17. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    Published
    March 17, 2016, 2:30 p.m.
    ...terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host machinery...gene start) and a transcription termination/polyadenylation signal sequence (gene end). A minigenome is...minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP encapsidates the negative...then recognizes the encapsidated minigenome as a template for replication and transcription, as it would... necessary control elements for each would be relatively large. Cloning the viral genes in this plasmid...-minigenome). The anti-minigenome is used as a template by the viral polymerase to synthesize additional...addition, the polymerase will use the minigenome as a template to transcribe the reporter gene (e.g. firefly ...

  18. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    Published
    Oct. 6, 2015, 2:30 p.m.
    ...location which can all be extrapolated from the 2D barcode and LIMS: Barcoded Tube Plate Freezer Rack Freezer... location within a plate that can hold up to 96 tubes (see image below). Each plate has numbers and letters... Each sample that is transformed is plated onto a petri dish plate that is also barcoded. The barcodes... to give coordinates, from A1 through H12. Each plate is also barcoded with a 10 digit barcode and then...rack that contains five levels that can hold five plates on each level. This rack is located in one of five...sample type. It is critical for us to know which plate, rack, and freezer each sample is located in. Otherwise...identifies the contents of each individual tube.  If a plate of samples is ever dropped, we can simply scan the...

  19. Using Video to Share Your Science: We Share Science

    Type
    Blog Post
    Published
    Nov. 3, 2016, 2:30 p.m.
    ...not hosted on the platform, they can be hosted on YouTube, Vimeo, and other platforms (such as university...economics, or maybe even biology or international relations.  Many of the challenges in science today are ...appealing to many audiences. While all of these platforms were developing, Google acquired Youtube and has...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...

  20. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...to the template strand. Therefore, I suggest that picking a crRNA complementary to the template strand...bound by a protein of interest are pulled-down and later coupled to high-throughput sequencing to identify...optimized to achieve high knock-in efficiency. To calculate the tagging efficiency in the transfected cells...tagging experiments by assembling csRNPs in a 96-well plate format and tested parallel tag knock-in for 96 different...single go (Figure 3). We were able to tag >30 genes/plate in the first shot with typical knock-in efficiencies...knock-in experiment were pre-assembled in a 96-well plate. Glioma stem cells were transfected in 96-well format...device; 5 days after the transfection, the entire plate was scanned for V5 tagging using an Operetta imaging...
Showing: 101 - 120 of 792 results