...Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (ideally AAV-293T clones...Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other transfection reagents may be used in this protocol, but their conditions... with PEI and allow for the addition of other components, such as sorbitol and sodium bicarbonate, sorbitol...

...(or other small flame source) Incubator Marker Reagents LB agar plate (with appropriate antibiotic) Bacterial...DNA or use the individual colonies for other experiments....

...burner (or other small flame source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic...will work for this assay, and the above result represents a single experiment. For publishable data, the...

...purification allows you to isolate and purify DNA fragments based on size. The procedure starts with standard...Gel Electrophoresis Protocol with the following amendments: Notes: Gel purification is most efficient with...

...different cell lines and different transfection reagents. Workflow Timeline Day 0: Seed HEK293T cells (...incubator Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-...

...alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for a variety...container pH meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-...

...multichannel pipette 20–200 µL multichannel pipette Reagents Universal SYBR Master Mix 2X Primer pair targeting...plots obtained from an AAV sample. Each curve represents a dilution. References Aurnhammer C, Haase M,...

.... Use lots of plants: Lots of plants produce lots of seeds. I typically use 8-10 plants per construct ...of non-transformants and make finding true transformants more difficult. Don’t leave your plants on selective...and Dinneny, 2013). Preparing plants for transformation Whatever plants you are transforming into will...efficiency will be low. Tips: Use healthy plants: Your plants should show no signs of stress, such as anthocyanin...recover a transformant. Use young plants: If you transform early, your plants will still have plenty of time...PCR and glycerol stocks. Dip/drip plants more than once: Dip your plants, let them recover for a week, and...integration. Selecting Arabidopsis transformants Once your seeds from your T0 plants are dry, it’s time to select...

... and purify the DNA fragments. Finally, you simply incubate these three fragments together along with ... region between fragments also better ensures correct assembly order of the fragments as compared to the...chromosome fragments. CRISPR cutting was used to circumvent the need to PCR amplify the fragments of the ...regions, have no requirements for restriction enzymes, and no scar sequences between fragments. Some of these...and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives to ...are using Gibson assembly to put together DNA fragments. As a fun way to start, I highly recommend watching...allowing easy assembly of multiple linear DNA fragments, but can also be used in basic cloning of an insert...

...Delivering CRISPR components to plant cells Once you have selected the correct CRISPR components for your application...deliver these components to your target cells. Remember, efficient delivery of CRISPR components is essential... editors have been developed for plants and prime editing for plants has been developed for rice by Yiping...used to efficiently deliver CRISPR-Cas9 components into plants. Nanoparticles (carbon nanotubes, Kwak ...the potential of CRISPR for genome engineering in plants is also being realized. There are a variety of ...using genome editing to change the genetic code of plants, including the development of crops with longer...requiring several rounds of selection to isolate plants with the phenotype of interest. Genome engineering...

...Successful editing of transformants then typically occurs in T0 plants (from callus) or T1 plants (Arabidopsis ...to betanin, a member of a class of pigments primarily found in plants in the order Caryophyllales, such... family than the red/purple plant pigments found in all other plants, anthocyanins.   Figure 1:...the development of the mutant plants, but the seeds needed for experiments can only be collected from heterozygous...heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore, are useful for research...with these mutant plants. Using RUBY to simplify the identification of these mutants will speed up my ...Antibiotic selection would only then kill the desired plants! PCR or fluorescent markers are currently used ...

...and recruit students to join their societies. Freshers Fairs tend to be used as welcome events for new students... students that provide ways for students to network, find groups with common interests, and settle into...their upcoming events and intro sessions, and give out useful items and information to students. Freshers ...information events and summer internship information sessions to help undergraduate students with their...develop a range of talks for both students and the general public, career events, and networking meetups. The...answer is probably no, but these fun and informative events are one of the main ways budding young scholars...members, who comprise undergraduates, graduate students, postdocs, faculty members, and the general public...

...companies, and governments on the most effective and efficient ways of delivering healthcare. Clients typically... that clients face imminently. Consulting firms deploy case teams, usually with 3-6 consultants, who work...biotech clients. In this realm, there are a few different kinds of challenges that consultants get called...therapeutics. Consultants often work with such teams to help make decisions about R&D investments in either...diseases they can treat, and diagnostic advancements. Consultants can help these teams develop communication...services that help diabetic patients manage doses and timings of their drug treatments. The process of rising...approach brings to patients, doctors, and insurance providers, and is crucial to how our clients’ products will...

...options for what reagents to use - organic solvents and detergents.  Organic Solvents  As mentioned above...antibodies. Detergents  Another common family of permeabilization reagents are detergents, such as Triton...these detergents avoid some of the negative effects caused by organic solvents. However, detergents can ...with this type of fixation*. Organic Solvents Organic solvents, generally acetone, methanol, or ethanol...benefits of both reagents - cross-linkers better preserve sample morphology, while organic solvents can reveal...your samples, the relationships between cellular components, and the accessibility of your target epitope...fixatives - chemical cross-linkers and organic solvents.  Chemical Cross-Linkers Chemical cross-linkers...

...individual variants found in tumors are exceedingly rare. As a result, how most of these “variants of unknown...scale laboratory-based functional experiments that classify which variants are likely gain-of-function, loss-of-function...Also, in principle, these experiments could find correlations between variants in a gene of poorly classified...revealed many tricks up their sleeves and most patients still don’t benefit from this approach. One central...cancer clinical sequencing report includes rare variants that haven’t been characterized? Some have developed...approach this challenge, using attributes of cancer variants such as evolutionary conservation, or predicted...hope researchers can use to characterize these variants in a high-throughput manner and therefore push...

...receptors) or with experiments on permeabilized cells. Biarsenical labeling reagents If you want to label...control experiments are needed to establish the dynamic range of your assay. These experiments include...pressing issues in your laboratory, then FRET experiments are a viable biophysical path to answers. What...lipid bilayers, organelles or other cellular impediments. FRET has been used in many applications, including...fluorophore delivered to the right place? FRET experiments require targeting the donor and acceptor fluorophores...experimental design is that the targeting specificity requirements are different for the donor and acceptor fluorophores... on the same street as the donor), then FRET measurements are possible. In fact, even if the majority ...

...ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments. Cas9 protein, annealed cr:tracrRNA...procurement of cr:tracrRNA reagents, we scaled up these tagging experiments by assembling csRNPs in a ...set up csRNP knock-in experiments If your lab is new to CRISPR/Cas9 experiments, there is no need to worry...CRISPR reagents (turnaround time 5-7 days); because the tracrRNA is the same in all csRNP experiments, it...proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by a protein of interest are pulled-down...adoption of CRISPR/Cas9 technology and rapid improvements in the field, it has recently become possible...CRISPR depends on successful delivery of three reagents to cells: a guide RNA, Cas9 protein, and a donor...

...creation process. Each module of Fragmid represents a class of components, such as your CRISPR enzyme or Pol...module, are the components. Using the handy website, users can simply select the components they want for...module fragments on the Fragmid website.   Step two will ask you to fill in module fragments from ...able to select components that work with your design. Note that you can browse all components by clicking...Fragmid doesn’t have the components I want? You can make your own Fragmid components! Ensure your backbone...’ terminus, and selection markers. Need fewer components? A spacer can be inserted instead, allowing everything...everything to stay in the correct order. Most Fragmid components are available for request on the Addgene website...

...quantitative data for regulatory elements, which has not been available for plants. Addgene: Will the MoClo tools...Remember plants. Every time you look at your plate, think about how much that’s on it are plants, and the...produce constructs and edit genomes so as to make plants, and agricultural crops in particular, resistant...elucidate the molecular interactions that go on between plants and their pathogens. As Patron explains it, her...been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus, or pathogens to their...have tested them to show that they work well in plants and give similar stability to transcripts as the...construct, you don’t have to worry about repeating elements anymore. The kit contains sequences for several...

...science teachers reached out to their students’ STEM-oriented parents looking for experts to come give guest...can recruit parents from any background, it really helps if activities are run by parents who can answer...groups of about 50 students each, and each group went on its own wonderful tangents. Some groups asked... the annual Math & Science Night, a chance for parents and other members of the community to share their...general meeting and a means to request funding for events, consider reaching out to it. PTAs have more flexibility...event-planning base and will know how to reserve space, time events, and network for volunteers. Speaking of volunteers...you live in a school district that has a lot of parents in STEM careers, chances are they’re wondering ...