Mentors at Your Benchside

#28 — SDS-PAGE gels can be run at constant current, constant voltage, or constant power. But which is best? Listen to this episode of Mentors at Your Benchside to discover the differences between current, voltage, and power, and how they affect how your gels run. Visit the original article for a summary table of running your SDS-PAGE gels at a constant voltage, current, or power. [1] We've also got tips and tricks for casting the perfect SDS-PAGE gel, [2] or read a refresher on how SDS-PAGE works. [3] Resources: 1. Constant Current or Voltage in SDS-PAGE: The Great Debate. Available at: https://bitesizebio.com/51744/constant-current-or-voltage-in-sds-page/. 2. A Simple SDS-PAGE Gel Recipe and 10-Step Casting Protocol for Perfect Gels. Available at: https://bitesizebio.com/59429/sds-page-gel-recipe/. 3. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/.

#27 — Optimal conditions for DNA ligation reactions are a delicate balance between DNA molecules interacting and the enzymatic ligation reaction. In this episode, you'll discover our top 6 DNA ligation tips to improve the efficiency of your ligations and increase your cloning success rate! Read the full article to get more practical advice for optimizing DNA ligation reactions for cloning. [1] Want more cloning advice? Read about Cloning with Compatible Cohesive Ends, [2] and discover how to Remove Unwanted DNA from Vectors. [3] Also, find out why T4 DNA ligase is such a popular ligase and perhaps the only ligation enzyme you'll ever need. [4] Resources: 1. DNA Ligation: How it Works & 6 Top Tips. Available at: https://bitesizebio.com/10279/how-dna-ligation-works/ 2. Assembling the Puzzle: Cloning with Compatible Cohesive Ends. Available at: https://bitesizebio.com/21716/assembling-the-puzzle-cloning-with-compatible-cohesive-ends/ 3. Tech Clinic #1: Removing Unwanted DNA from Vectors — beat Murphy's Law. Available at: https://bitesizebio.com/10228/removing-unwanted-dna-from-vectors-how-to-beat-murphys-law/ 4. T4 DNA Ligase: The Only Ligase You'll Ever Need? Available at: https://bitesizebio.com/46482/t4-dna-ligase-the-only-ligase-youll-ever-need/

#26 — Cryo-EM is a revolutionary imaging method that lets us see complex biostructures at higher and higher resolutions. But do you understand the mind-blowing science behind this technique? And what is cryo-electron microscopy, anyway? Why is the cryogenic aspect important, and how did it seemingly go from nothing to the big time? In the latest episode of Mentors At Your Benchside, we answer all of these questions and more! Check out the corresponding online article to access loads of follow-up resources to deepen your understanding of this topic.[1] Also, check out our related articles covering crucial sample preparation considerations for cryo-EM and its history from obscure to Nobel Prize winner. [2,3] Resources: 1. What Is Cryo-Electron Microscopy? Available at: https://bitesizebio.com/62871/what-is-cryo-electron-microscopy/ 2. Cryo-EM Sample Prep: 5 Crucial Considerations. Available at: https://bitesizebio.com/62619/cryo-em-sample-prep/ 3. A Short History of Cryo-Electron Microscopy: Available at: https://bitesizebio.com/62839/history-of-cryo-electron-microscopy/

#25 — Phenol extraction of DNA is a commonly used method for removing proteins from nucleic acids; for example, to remove proteins from cell lysate during genomic DNA preparation. It's commonly used, but not well understood. Listen to this episode to get a quick explanation of how phenol extraction of DNA works. Visit the original article to see handy diagrams on how phenol extraction works. [1] To get more help and advice on DNA extraction, read our related articles and discover how chloroform can clean up your phenol extractions, [2] learn how ethanol precipitation works, [3] and decide if ethanol or isopropanol is best for precipitating your samples. [4] Resources: 1. The Basics: How Phenol Extraction of DNA Works. Available at: https://bitesizebio.com/384/the-basics-how-phenol-extraction-works/ 2. Practical Application of Phenol-Chloroform Extraction. Available at: https://bitesizebio.com/3651/practical-application-of-phenol-chloroform-extraction/ 3. Ethanol Precipitation of DNA and RNA: How it Works. Available at: https://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/ 4. DNA Precipitation: Ethanol vs. Isopropanol. Available at: https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/

#24 — How can you best prepare for a postdoc interview? Here we cover 10 questions that are often encountered during the job-hunting process. Preparing answers to these questions, alongside doing some background research about the research group and PI, can give you the edge in securing that postdoc. You can recap these top 10 questions anytime by visiting the full article on popular postdoc interview questions. [1] If you are looking for further interview advice, make sure you check out our top 10 interview tips, consider how you might concisely describe your research so far, and get advice on what to wear for the interview. Resources: 1. Postdoc Interview Preparation: Sample Questions and Answers. Available at: https://bitesizebio.com/10393/postdoc-interview-preparation-sample-questions-and-answers/ 2. 10 Great Tips To Make A Good Impression At Your Interview. Available at: https://bitesizebio.com/6696/10-great-tips-to-make-a-good-impression-at-your-interview/ 3. Can You Describe Your Research in 30 Seconds? 60? Available at: https://bitesizebio.com/13639/can-you-describe-your-research-in-30-seconds-60/ 4. GOT AN INTERVIEW! What to wear? What to wear? Available at: https://bitesizebio.com/6261/got-an-interview-what-to-wear-what-to-wear/

#23 — Creativity in Science: How a Good Imagination Can Help Your Research If you're stuck in a research rut in the lab, developing your creativity can help you find new solutions to current problems that may be impeding your scientific progress. In this episode, we explore 3 important questions you should consider to help spark your creativity in the lab and provide 8 simple tips to help achieve this. Read the full article on boosting your creativity in science. [1] If you are looking for further advice, make sure you listen to our Happy Scientist Podcast episode on Imagination, which covers even more ways to boost your imagination and creativity. [2] Consider the importance of allowing yourself time to think and reflect. [3] Finally, listen to this episode of the Microscopists featuring Eric Betzig, where the Nobel Laureate reveals how his biggest ideas came during his two periods of unemployment. [3] Resources: 1. Creativity in Science: How a Good Imagination Can Help Your Research. Available at: https://bitesizebio.com/63742/creativity-in-science/ 2. Episode 33 — How to Foster Imagination. Available at : https://bitesizebio.com/podcast/episode-33-how-to-foster-imagination/ 3. Time to Think. Available at: https://bitesizebio.com/146/time-to-think/ 4. Eric Betzig (University of California). Available at: https://bitesizebio.com/podcast/eric-betzig/

#22 — Research often requires you to use dangerous chemicals. From caustic acids and bases to pH solutions and toxic reducing agents, chemical hazards abound in the lab. In this episode, we'll take a look at ten dangerous chemicals in the lab and the harm they can cause you. Want to develop your understanding of chemical safety? Why not read the full article, [1] where you'll find lots of helpful resources and further reading? If you fancy a deeper dive into all aspects of lab safety, then check out the Bitesize Bio lab safety eBook. [2] If you're a bit rusty on your good laboratory practice, definitely read 10 Stupid Lab Safety Mistakes [3] and see how many sins you commit. And finally, we all rely on our PPE for protection and safety. Be sure to take a look at our 10 Common PPE Sins for a quick brush-up. [4] Resources: 1. Ten Bad Chemicals in the Lab and What They do to You! Available at: https://bitesizebio.com/10470/ten-bad-chemicals-in-the-lab-and-what-they-do-to-you/ 2. The Bitesize Bio Guide to Lab Safety eBook. Available at: https://bitesizebio.com/wp-content/uploads/2021/08/The-Bitesize-Bio-Guide-to-Lab-Safety.pdf 3. 10 Stupid Lab Safety Mistakes. Available at: https://bitesizebio.com/1899/stupid-lab-safety-mistakes/ 4. 10 Common PPE Sins. Available at: https://bitesizebio.com/9850/10-common-ppe-sins/

#21 — Do you know the difference between cell confluency and cell number? Can you measure cell confluency accurately? Cell confluency measurements are essential in cell-based experiments. Listen to this episode to learn what cell confluency is and the different quantification methods to measure it. Read the original article to get visual guides to help you understand and measure your cell confluency accurately. [1] Do you want more information to help you perfect your cell culture? Check out related articles to learn how to get the right passage number for your cells and how to culture adherent cells like HEK293s. [2,3] Resources: 1. How Confluent Are Your Cells? A Beginner's Guide to Measuring Cell Culture Confluency. Available at: https://bitesizebio.com/63887/cell-confluency/ 2. What's in a Number: Getting the Right Passage in Cell Culture. Available at: https://bitesizebio.com/13685/cell-culture-passage-number-explained/ 3. What the HEK? A Beginner's Guide to HEK293 Cells. Available at: https://bitesizebio.com/45489/guide-to-hek293-cells/

#20 — Have you ever wondered how Laemmli buffer actually works? In this episode of Mentors At Your Benchside, we talk through the different components of Laemmli buffer, what they do and why they are essential for your SDS-PAGE experiments. Read the full article to learn more about this buffer and get a handy Laemmli buffer recipe. [1] Looking for more information on SDS-PAGE? Discover the theory behind SDS-PAGE and get advice on how you can cast the perfect SDS-PAGE gel. [2,3] You can also download our useful SDS-PAGE protocol PDF that contains simple buffer recipes, gel recipes, and a neat casting protocol. [4] Resources: 1. Laemmli Buffer: What Is It for Anyway? Available at: https://bitesizebio.com/44540/laemmli-buffer-what-is-it-for-anyway/ 2. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/ 3. A Simple SDS-PAGE Gel Recipe and 10-Step Casting Protocol for Perfect Gels. Available at: https://bitesizebio.com/59429/sds-page-gel-recipe/ 4. Bitesize Bio's SDS-PAGE Protocol PDF Cheat Sheet. Available at: https://bitesizebio.com/sds-page-protocol-pdf/

#19 — Listen to this episode to discover what alternative splicing is, the history of how it was discovered, and get key examples of splicing in action. Read the full article to learn more about alternative splicing, including a visual diagram of how splicing works. [1] Read our related articles to find out how alternative splicing can affect your experiments and discover methods for detecting splice variants. [2,3] Resources: 1. What is Alternative Splicing, and Why Is It Important? Available at: https://bitesizebio.com/10148/what-is-alternative-splicing-and-why-is-it-important/ 2. How Can A Single Mutation Affect Splicing Regulation? Available at: https://bitesizebio.com/10307/how-can-a-single-mutation-affect-splicing-regulation/ 3. How to Detect Alternative Splicing Variants. Available at: https://bitesizebio.com/10138/how-to-detect-alternative-splicing-variants/

#18 — Why are SDS-PAGE gels run vertically? What would happen if we ran them horizontally? Has anyone ever tried? Discover the answers to these questions and more in this episode of Mentors At Your Benchside. To see what others are saying and join in the conversation, head over to the comments section in the original article on why SDS-PAGE is run vertically. [1] If this episode has ignited your desire for answers, Check out some of our related articles to learn the how SDS-PAGE works and discover whether running your SDS-PAGE gels with constant current or constant voltage is best. [2,3] Resources: 1. Why Is SDS-PAGE Run Vertically? Here are 3 Great Answers. Available at: https://bitesizebio.com/10699/why-is-sds-page-run-vertically/ 2. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/ 3. Constant Current or Voltage in SDS-PAGE: The Great Debate. Available at: https://bitesizebio.com/51744/constant-current-or-voltage-in-sds-page/

#17 — Why do some plasmids give a better protein yield than others? It may have a lot to do with the plasmid copy number. In this episode, we talk about what plasmid copy number means, why it is important and how you can manipulate it to get the most out of your experiments. To learn more about plasmid copy number, read the full article on our site. [1] Resources: 1. https://bitesizebio.com/22824/how-to-manipulate-plasmid-copy-number/

#16 — Is your viva on the horizon? Many people think viva exams are going to be painful. But what if you could make your viva go smoothly and maybe even enjoy it? Listen to our top 10 viva tips to get advice from a viva survivor. To learn more about surviving your viva, read the full article on our site. [1] Resources: 1. https://bitesizebio.com/10109/top-10-tips-for-viva-success/

#15 — In the last few years, CRISPR-Cas9 has become a popular genome editing tool, but have you ever wondered where it came from? In this episode, we explore the history of CRISPR-Cas9, from the first CRISPR discovery in 1987 to current applications in targeted genome and epigenome editing. Read the full article to learn more about the history of CRISPR-Cas9 and to see a timeline of discoveries. [1] Resources: 1. https://bitesizebio.com/47927/history-crispr/

#14 — Do you know how accurate and precise your measurements are? Imprecise and inaccurate measurements can have a dramatic impact on your results. Listen to get 8 top tips for making your measurements more accurate and precise. To learn more about accuracy and precision, read the full article on our site. [1] Resources: 1. https://bitesizebio.com/55470/accuracy-and-precision/

#13 — There are now hundreds of different tailored fluorescent proteins to choose from that can improve your experiments in the lab. What are the key considerations when choosing a fluorescent protein for your experiment? Should you consider using a fancy new fluorescent protein? Where can you find the best resources to help you out? Find out the answers to these and more in this episode of Mentors At Your Benchside. Please read the full article, which includes our super helpful table of fluorescent proteins and their properties. [1] Resources: 1. https://bitesizebio.com/54287/choosing-a-fluorescent-protein/

#12 — As a follow-up to our episode about ethanol precipitation of DNA and RNA, [1] this episode explains the differences between DNA precipitation in ethanol and isopropanol, helping you to figure out which method is the best choice for your experiment. Read the full article for handy protocol tips, the differences between using ethanol and isopropanol, and when to use each method. [2] Resources: 1. https://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/ 2. https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/

#11 — Fluorescence is undoubtedly one of the most important and useful tools in a biologist's toolbox. But do you actually know how fluorescence works? In this episode, discover what the three steps of fluorescence are, and how fluorescence can be used in flow cytometry. Read the full article for a breakdown of the key points of fluorescence. [1] Resources: 1. https://bitesizebio.com/32973/fluorescent-molecules/

#10 — As you probably know, there are different types of ethanol found in biology labs. It's a versatile solvent used in dozens of experiments and procedures, including disinfection, DNA precipitation, and tissue dehydration. But which type of ethanol should you use for your application? What's the difference between the types of ethanol in your lab? And how do you handle it appropriately? In this episode, we answer these questions, providing you with the information you need to keep your research producing top-notch results. Read the full article on Which Type of Ethanol You Should Use for links to additional resources on laboratory applications of ethanol. [1] Resources: 1. https://bitesizebio.com/13518/which-type-of-ethanol-should-i-use/

#9 — One of the most commonly used cell lines in molecular biology labs is the Human Embryonic Kidney HEK293 cell line. Listen to this episode to find out all about the history of HEK293 cells and how to work with them. Read the full article for more details about working with HEK293 cells. [1] Resources: 1. https://bitesizebio.com/45489/guide-to-hek293-cells/

#8 — You probably have or will use SDS-PAGE at some point to separate proteins, but do you really understand how this technique works? Knowing how SDS-PAGE works means you can tweak and troubleshoot your technique as well as impress your supervisor and lab mates. In this episode, we take you through how SDS-PAGE works, including what SDS does, why you need to use a reducing agent like DTT or beta-mercaptoethanol, and the critical importance of the stacking gel. Read the full How SDS-PAGE Works article to see helpful visuals for how this technique works and access the table showing the protein sizes that different acrylamide percentages can separate. [1] Expand your knowledge by buffing up on Laemmli buffer and get our Guide to Gradient Gels. [2,3] If you pour your own SDS-PAGE gels, take a deep dive into the chemistry of how gels work and learn how to pour perfect gels every time with our Simple SDS-PAGE Gel Recipe with 10-Step Casting Protocol. [4] Resources: 1. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/ 2. Laemmli Buffer: What Is It for Anyway? Available at: https://bitesizebio.com/44540/laemmli-buffer-what-is-it-for-anyway/ 3. A Guide to Gradient Gels: The Why's and How's. Available at: https://bitesizebio.com/47184/gradient-gels/ 4. A Simple SDS-PAGE Gel Recipe and 10-Step Casting Protocol for Perfect Gels. Available at: https://bitesizebio.com/59429/sds-page-gel-recipe/

#7 — In this episode, we cover how jellyfish were able to have a huge impact on biology through the discovery and development of GFP. Using GFP is now ubiquitous in pretty much all fields in biology, and we take you through how three Nobel Laureates developed this valuable research tool. [1] Many of the applications for GFP are microscopy-based, and we discuss how you can utilize GFP for translational and transcriptional fusions, FRAP, FLIP and FRET experiments in the lab. [2,3] Read the full article for more useful links, hints, and tips on using GFP in your experiments. [4] Resources: 1. Press release. NobelPrize.org. Nobel Prize Outreach AB 2022. Fri. 1 Jul 2022. Available at: https://www.nobelprize.org/prizes/chemistry/2008/press-release/ 2. Fun With FRAP! Fluorescence Recovery After Photobleaching for Confocal Microscopy. Available at: https://bitesizebio.com/19946/fun-with-frap-fluorescence-recovery-after-photobleaching-for-confocal-microscopy/ 3. Using Flow Cytometry for Fluorescence Resonance Energy Transfer. Available at: https://bitesizebio.com/21935/using-flow-cytometry-for-fluorescence-resonance-energy-transfer/ 4. How a Jellyfish Changed Biology: the Discovery and Development of GFP. Available at: https://bitesizebio.com/13390/gfp/

#6 — Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acid preparations in an aqueous solution. In this episode, we'll bring you up to speed on how ethanol precipitation works, including the importance of solubility, the roles of salt, ethanol, and temperature, plus a few helpful tips on the side. Read the full article to learn more about the ins and outs of ethanol precipitation and other DNA clean-up approaches. https://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/

#5 — Have you ever chilled beer on dry ice from the lab? Does your wardrobe consist mainly of free T-shirts from companies at conferences? (https://bit.ly/conference-who-where-what) Have you ever wondered what the distilled water in the lab tastes like? (https://bit.ly/water-what-is-it-good-for) If any of these questions apply to you, then tune in to this episode, as we cover more funny and telling signs that you're a true scientist. Read the full article to discover the difference between a Science Fan and a bona fide Scientist - https://bitesizebio.com/36753/20-telling-signs-scientist/

#4 — PCR is a useful lab technique used to copy, sequence or quantify DNA. If you're new to PCR, this episode is for you. We cover the five things you need to get started with PCR and explain how to run a polymerase chain reaction. Read the full article to learn more about setting up a PCR. [1] Learn more about Kary Mullis and the invention of PCR, and about the first thermocycler "Mr Cycle". [2–4] For practical tips, see our article on the top tips for primer design. [5] Resources: 1. What is PCR? The Beginner's Guide. Available at: https://bitesizebio.com/19132/pcr-basics-what-is-pcr/ 2. Kary B. Mullis – Facts. NobelPrize.org. Nobel Prize Outreach AB 2022. Fri. 1 Jul 2022. Available at: https://www.nobelprize.org/prizes/chemistry/1993/mullis/facts/ 3. The Invention of PCR. Available at: https://bitesizebio.com/13505/the-invention-of-pcr/ 4. Mr. Cycle, Thermal Cycler. Available at: https://americanhistory.si.edu/collections/search/object/nmah_1000862 5. How to Survive a Difficult PCR. Available at: https://bitesizebio.com/37205/how-to-survive-a-difficult-pcr/

#3 — In this episode of Mentors At Your Benchside, listen to a short history of cryo-electron microscopy—the Nobel Prize-winning technique revolutionizing molecular and structural biology. The journey takes us from the inception of cryo-EM- obscure, inferior, and derided- to the present-day competition for access to the incredibly powerful Krios microscopes. It's all about the power of an image. And speaking of images, be sure to read A Short History of Cryo-Electron Microscopy for a graphical timeline and some stunning cryo-EM structures. [1] Fascinating, cross-disciplinary science underpins cryo-EM—the technique is as broad as it is popular. To expand your understanding of the fundamental science behind it, Read What Is Cryo-Electron Microscopy? A Brief Introduction. [2] For tips and tricks on getting your sample ready for a cryo-EM experiment, check out Cryo-EM Sample Prep: 5 Crucial Considerations. [3] And for a simple illustration of the stunning structures cryo-EM produces, browse the Electron Microscopy Data Bank. [4] Resources: 1. A Short History of Cryo-Electron Microscopy. Available at: https://bitesizebio.com/62839/history-of-cryo-electron-microscopy/ 2. What Is Cryo-Electron Microscopy? A Brief Introduction. Available at: https://bitesizebio.com/62871/what-is-cryo-electron-microscopy/ 3. Cryo-EM Sample Prep: 5 Crucial Considerations. Available at: https://bitesizebio.com/62619/cryo-em-sample-prep/ 4. Electron Microscopy Data Bank. Available at: https://www.ebi.ac.uk/emdb/

#3 — In this episode of Mentors At Your Benchside, listen to a short history of cryo-electron microscopy—the Nobel Prize-winning technique revolutionizing molecular and structural biology. The journey takes us from the inception of cryo-EM- obscure, inferior, and derided- to the present-day competition for access to the incredibly powerful Krios microscopes. It's all about the power of an image. And speaking of images, be sure to read A Short History of Cryo-Electron Microscopy for a graphical timeline and some stunning cryo-EM structures. [1] Fascinating, cross-disciplinary science underpins cryo-EM—the technique is as broad as it is popular. To expand your understanding of the fundamental science behind it, Read What Is Cryo-Electron Microscopy? A Brief Introduction. [2] For tips and tricks on getting your sample ready for a cryo-EM experiment, check out Cryo-EM Sample Prep: 5 Crucial Considerations. [3] And for a simple illustration of the stunning structures cryo-EM produces, browse the Electron Microscopy Data Bank. [4] Resources: 1. A Short History of Cryo-Electron Microscopy. Available at: https://bitesizebio.com/62839/history-of-cryo-electron-microscopy/ 2. What Is Cryo-Electron Microscopy? A Brief Introduction. Available at: https://bitesizebio.com/62871/what-is-cryo-electron-microscopy/ 3. Cryo-EM Sample Prep: 5 Crucial Considerations. Available at: https://bitesizebio.com/62619/cryo-em-sample-prep/ 4. Electron Microscopy Data Bank. Available at: https://www.ebi.ac.uk/emdb/

#2 — Micropipettes are the bread and butter instrument of a lab, helping you to accurately dispense minute amounts of liquid. But these tools are only trustworthy if they are well maintained and handled appropriately. Listen to the latest episode of Mentors At Your Benchside to uncover top tips for precise pipetting. Want to know more about correct pipetting technique? Access the original article on 17 Ways to Avoid Pipetting Errors, download the Gilson Guide to Pipetting, and read the Nature article showing the effect of sample temperature on pipetting volume. [1–3] Resources: 1. 17 Ways to Stop Pipetting Errors From Ruining Your Experiments. Available at: https://bitesizebio.com/344/17-ways-to-stop-pipetting-errors-ruining-your-experiments/ 2. Gilson Guide to Pipetting. Available at: https://gb.gilson.com/GBSV/guide-to-pipetting 3. Millet, F., Barthlen, T. Securing accuracy and precision when pipetting hot and cold liquids with Microman®. Nat Methods 4, iii–iv (2007). https://doi.org/10.1038/nmeth1086

#1 — Is it worth doing a PhD? This is a question that probably plagues every research student at some point in their career. In this episode, we explore 5 important questions you should consider before embarking on a PhD. Read the full article for more advice before making the difficult decision to pursue a PhD after getting your Master's degree. [1] If you are looking for further advice, make sure you check out our article with pointers for PhD students. [2] A good PhD supervisor is worth their weight in gold and finding a good mentor should be a priority. [3] And, if you're sure that a PhD is the right move for you, then search for PhDs in Biological and Medical Sciences to find the right PhD to suit you. [4] Finally, read our handy article that lists some alternative career options for scientists. [5] Resources: 1. Is it Worth Doing a PhD After a Master's? Available at: https://bitesizebio.com/11022/is-it-worth-doing-a-phd-after-a-masters/ 2. 10 Do's and Don'ts for PhD Students. Available at: https://bitesizebio.com/articles/10-dos-and-donts-for-phd-students/ 3. Picking an Advisor: The Good, The Bad, and The Ugly. Available at: https://bitesizebio.com/articles/picking-an-advisor-the-good-the-bad-and-the-ugly/ 4. Find a PhD. Available at: https://www.findaphd.com/phds/biological-and-medical-sciences/?10gc00 5. Alternative Careers For Scientists. Available at: https://bitesizebio.com/301/alternative-careers-for-scientists/

Although macrophages were first described by Elie Metchnikoff in 1882, plenty of mysteries are still associated with the cell type. Indeed, while macrophages were once considered simply "garbage trucks" of the immune system due to their phagocytic property, their substantial and multifaceted contribution to immunological responses and homeostasis is becoming more apparent. Macrophages can produce a wide range of cytokines and chemokines to influence the immune response toward healing or inflammation. In as such, they possess a great deal of plasticity to respond with either pro- or anti-inflammatory signals depending on the environmental milieu. Moreover, researchers are beginning to turn to macrophages to assist chimeric antigen receptor (CAR) T cells in various immunotherapies. This webinar is presented by Anne Lodge, Chief Scientific Officer of Astarte Biologics.

Functional imaging is a rapidly growing field key to driving new understanding in biology. Insights into the function and interaction of molecules are the key to reveal the underlying cellular mechanisms. In this context, fluorescence lifetime imaging (FLIM) is a powerful tool, providing valuable information beyond spectral imaging. FLIM is immune to concentration artefacts and sensitive to molecular environment such has pH changes, ion concentrations, and more. Förster Resonance Energy Transfer (FRET) is an example of molecular environmental changes. The donor lifetime is shortened by the presence of the acceptor. FRET experiments thus benefit from FLIM information. The FRET-FLIM readout is independent of the donor or acceptor concentration making it the quantification assay of choice. Recently, FRET has been exploited to engineer sensors (FRET biosensors). FRET signal changes in these biosensors correspond to either binding or release of a ligand. How this type of readout can help uncover biological mechanisms was recently shown in a Nature Immunology article (Anzilotti 2019). The approach used in this article, and described in this webinar, opens the field to imaging in a range of situations in primary cells, where sensitivity and the need to avoid damaging the cells are both paramount.

In this tutorial on confocal imaging, you will learn how you can: - Capture weaker signals—and still get sound, reproducible data. - Reach faster volumetric imaging without sacrificing resolution - Increase data throughput for your imaging needs Learn about the new Multiplex mode for parallel pixel acquisition with the ZEISS LSM 9 family and Airyscan 2. You can now acquire up to 8 superresolution image lines with high signal-to-noise rate in a single sweep. Capture dynamic processes, cellular signaling, molecular trafficking, and diffusion events with real-time superresolution and high SNR. The new Multiplex mode for Airyscan 2 uses smart illumination and detection schemes for parallel pixel acquisition on a confocal microscope. Scientists can now capture weaker signals, keep their context, and get statistically sound data. Extending Airyscan imaging to larger model systems with lower expression levels, the new Multiplex mode increases acquisition speeds even further. You get superresolution and a 4 times higher SNR compared to traditional confocals. This novel concept allows rapid volumetric imaging with unprecedented resolution beyond what is available in traditional confocal systems today. Airyscan 2 provides new data handling concepts, providing 6.6 times smaller data sizes and 5 times faster image reconstruction times. Further, optimized real time acquisition strategies employed with the LSM 9 family enable faster scan speeds for Airyscan 2, allowing higher data throughput.

In this webinar, you will learn: - The complete workflow for in situ cryo-electron tomography - How subtomogram averaging within the cell yields native-state structures of macromolecular complexes (e.g., the asymmetric and dilated nuclear pore of algae) - How mapping these structures back into the native cellular environment reveals new molecular interactions that are only accessible by this technique (e.g., the binding of cargo to COPI-coated Golgi membranes and the tethering of proteasomes to the nuclear pore). Cryo-electron tomography can visualize macromolecular structures in situ, inside the cell. Vitreous frozen cells are first thinned with a focused ion beam and then imaged in three dimensions using a transmission electron microscope. This transformative method has the power to revolutionize our understanding of cell biology, revealing native cellular architecture with molecular clarity.

In this webinar, you will learn: - How structural biology can change the face of your research - The biological challenges of cancer research - Advantages of cryo-EM for your research Covering both biology and methodology, this webinar will explain how single-particle cryo-electron microscopy enables us to gain insight into cancer development through the detailed analysis of molecular structure. In single-particle cryo-EM, hundreds of thousands of images formed by electron scattering of individual molecules or complexes are analyzed to derive their three-dimensional structure. Technological and computational advances have dramatically transformed the field of cryo-EM in the past years, enabling structural insights at near-atomic resolution into assemblies that had not been tractable using any other structural biology technique. Consequently, cryo-EM has become a mainstream method structural biology, with a multitude of new facilities and research groups being established all over the world within just a couple of years. This webinar will address the roles that structural biology has been playing in cancer research, uncovering cellular processes involved in cancer development and protection, and guiding drug discovery efforts. The biological challenges of cancer research will be discussed, as well as the unique strengths of cryo-EM as an experimental approach towards these questions, briefly covering the methodology and procedures in sample preparation and data processing. We will illustrate these aspects with some of the latest research from the laboratory of Eva Nogales at UC Berkeley and assess the promises and challenges of cryo-EM in our fight against cancer.

Your life sciences research often requires you to measure, quantify and understand the finest details and sub-cellular structures of your sample. You may be working with tissue, bacteria, organoids, neurons, living or fixed -cells and many different labels. In this webinar, we will explain how Elyra 7 with Lattice SIM takes you beyond the diffraction limit of conventional microscopy to image your samples with superresolution. Learn how to examine the fastest processes in living samples – in large fields of view, in 3D, over long time periods, and with multiple colors. The new Lattice SIM technology of Elyra 7 brings structured illumination microscopy (SIM) to a new level. Groundbreaking light efficiency gives you gentle superresolution imaging with incredibly high speed – at 255 fps you will get your data faster than ever before. See how Elyra 7 lets you combine Lattice SIM with single molecule localization microscopy (SMLM) for techniques such as PALM, dSTORM and PAINT. Choose freely among your labels when imaging with resolutions down to 20 nm laterally. High power laser lines allow you to image your sample with ease, from green to far red. Elyra 7 is also very flexible: you can employ a wealth of contrasting techniques and combine them with optical sectioning. The new Apotome mode gives you superfast optical sectioning of your 3D samples. All that, plus Elyra 7 works seamlessly with your ZEISS SEMs in a correlative workflow.

Array tomography (AT) is a 3D image reconstruction technique for high resolution, quantitative analysis of biological structures. For optimal results, ultrathin and ordered sections are an absolute requirement. In this webinar you will get tips and tricks to optimize the workflow of your array tomography: - Fast and precise trimming of the sample block-face - Adhesion of a single section to create ribbons - Automated serial sectioning with the ARTOS 3D ultramicrotome - Acquisition and processing of a 3D SEM dataset - Segmentation and 3D reconstruction of cells - Interpretation of 3D reconstructions

In this webinar you will learn: - Improved vitrification: Specimen vitrification without synchronisation fluid - Vitrification strategies: Optimized freezing for different techniques - Light and electrical stimulation: Dissect cellular processes with millisecond precision - A look into the future: freezing of crystals Plunge freezing and cryo imaging of proteins and complexes have revealed new details in understanding the machinery of the cell and how molecules are involved in cellular processes. However, most eukaryotic cells and tissue samples cannot be plunge frozen because of the rapid decay of the cooling rate within the sample during freezing. High pressure freezing, on the other hand, is currently the main approach to vitrify larger samples (up to 200 µm) and to capture the intrinsic changes in fine structure or cellular dynamics. To further improve its cryo solutions, Leica developed a new cryo platform: the EM ICE. This new generation cryo platform combines speed, reliability and flexibility to facilitate research in various scientific fields. The EM ICE allows users to freeze samples within milliseconds and even permits the combination of high pressure freezing with optogenetics and electrophysiology.

Join Dr. Jason Reed as he describes a novel method by which endonuclease-inhibited Cas9 can be employed as a programmable biomarker in high-speed atomic force microscopy (HS-AFM) imaging. In this webinar, you will learn: 1. How CRISPR/Cas9 can be used to "flag" alterations and mutations in DNA, rather than cut it 2. How pairing atomic force microscopy (AFM) with optical equipment found in DVD players can be used to map DNA at a faster rate than traditional DNA sequencing 3. Applications of this technology, particularly in relation to discovering and diagnosing genetic diseases Since the diameter of the Cas9 molecule is greater than that of DNA, they are easy to locate along the DNA strand. Taking advantage of this, Jason's lab reported approximately 90% Cas9 binding accuracy to DNA molecules under optimized conditions. The alignment of single-molecule maps with nanoscale resolution becomes far more computationally straightforward than if labels are localized with multi-kb ambiguity. This process yields reduced processing time and cost for assembling a consensus map. Given its single-molecule sensitivity, approximately 15 bp accuracy, and no amplification requirement, Dr. Reed's novel method is amenable to small sample sizes. This proves to be an advantage in clinical situations where obtaining the almost 10 μg of DNA required for single-molecule sequencing is extremely difficult—if not impossible!

Join Theo Roth as he describes his lab's novel CRISPR-Cas9 genome-targeting system that does not require viral vectors to modify T cell genomes, but instead focuses on HDR. This allows rapid and efficient insertion of large DNA sequences at specific sites in the genomes of primary human T cells, and permits individual or multiplexed modification of endogenous genes. Importantly, avoiding the use of viral vectors will result in accelerated research and clinical applications, reduce experimental cost, and improve safety. In this webinar, you will learn: - The advantages of using HDR versus recombinant viral vectors when modifying T cell genomes - How long double-stranded and single-stranded DNA can serve as a non-viral HDR template - A novel method that allows for the insertion of large DNA sequences (>1Kb) without a virus! Current efforts at reprogramming T cells for therapeutic purposes rely on using recombinant viral vectors. Unfortunately, viral vectors do not target transgenes to specific genomic sites. Moreover, the manufacturing and testing of effective viral vectors is often a lengthy and expensive process, which slows research progress and clinical use. However, recent studies have shown that re-engineering T cells in a specific and efficient manner is possible using homology-directed repair (HDR).

Join us in this webinar as Dr. Victoria Doronina helps you determine the quality of your nucleic acids. In this webinar you will learn: - How to choose the best method to extract your nucleic acids - Which method you should chose to determine nucleic acid quality - How to avoid common pitfalls for both extraction and quality control Virtually all experiments in the molecular biology lab require high quality, pure nucleic acids as a starting material. This sounds simple enough. However, as always, the trouble is in the details. There are several methods to isolate your nucleic acids. But which one is best for your experiment? How do you determine nucleic acid quality? And why does it seem to not be working? Watch this webinar to find out the answers. Victoria will breakdown the nucleic acid purification process and how to match the right method to the right type of nucleic acid. She will also show you how to avoid common pitfalls and contaminants in isolation and quality control. This webinar is essential for anyone in a molecular biology lab who wants better results in downstream experiments!

In this webinar, you will learn: - How to best prepare your specimen for live cell isolation by laser microdissection - How to optimize your laser microdissection workflow - How to avoid common pitfalls of this technique Laser microdissection is a tool for the isolation of homogenous cell populations from their native niches in tissues to downstream molecular assays. Beside its routine use for fixed tissue sections, laser microdissection may be applied for live cell isolation. Unlike other well-established and widely used techniques for live cell isolation and single cell cloning—such as FACS, MACS, cloning by limited dilution, and so on—laser microdissection allows for capturing live cells and cell colonies without their detachment from the carrier. In other words, there is no need to prepare a single cell suspension before the isolation procedure using mechanical and enzymatic dissociation, which can affect cell fate after plating. This feature of laser microdissection is desirable for stem cell research. We established a simple strategy for the efficient live cell isolation using the Leica Laser Microdissection platform. We were able to demonstrate not only colony formation from the isolated samples containing live cells, but also single cell cloning. In this webinar, specimen preparation, laser adjustment, overall workflow, and limitations on live cell isolation by laser microdissection are discussed.

In this webinar, you will learn: - How to overcome issues with stage drift and crosstalk between channels - How to obtain reliable images and reproducible quantification results - How to proceed with advanced image analysis after restoring images During fluorescence image acquisition, many experimental uncertainties are introduced that affect the correct object interpretation and analysis. The blurring and the noise implicit in the image formation are two of the largest sources of experimental trouble. Additional aberrations, such as stage drift, and crosstalk and chromatic aberration between channels, can also affect the imaging. Huygens image deconvolution and restoration is a proven method to revert these issues and recover a more realistic representation of the original object. After restoration of the image, you can proceed with the advanced Huygens analysis options for colocalization, object measurements, and tracking. This webinar will illustrate how you can make optimal use of the complete Huygens workflow to obtain reliable images and reproducible quantification results, focusing on the advanced analysis options offered by the Huygens software.

Join us in this webinar featuring Dr. Omonse Talton who will guide you through developing a "fool proof" enzyme-linked immunosorbent assay (ELISA). In this webinar, you will learn: - When and why you should use the different types of ELISAs– direct, indirect, sandwich, competitive/inhibition ELISA - Major considerations for developing your ELISA - Tried and tested tips for you to perform a successful ELISA The ELISA is one of (if not the most) common techniques in biology and biochemistry laboratories. You can use an ELISA to detect minute amounts of protein for medical diagnostics, for testing food for common allergens, and in toxicology screens screen for certain drugs. While the overall premise of an ELISA is simple, as with any assay, its success hinges on the experimental set up. In this webinar, Omonse will give you a crash course on that experimental set up—from start to finish. She will discuss the different types of ELISAs, the major considerations in development of the assay, and troubleshooting—including tips and tricks. With this webinar, you will be able to ensure that your ELISA provides reproducible and publishable results!

In this webinar, Dr. Stephen Pettitt explains how he applies genome-wide targeted mutagenesis screens to elucidate the genetic basis of drug resistance. Using mouse and breast cancer cell lines, Dr. Pettitt’s team developed a targeted, genome-wide mutagenesis screen to identify mutations responsible for resistance to the potent PARP inhibitor talazoparib (BMN 673). The screen yielded one particularly interesting point mutation in the PARP1 gene. This mutation disrupted the ability of PARP1 to bind DNA, demonstrating that DNA binding is necessary for the action of talazoparib. Dr. Pettitt will describe how he then employed a high-density, focused sgRNA library targeting PARP1 to generate further mutants that he used to elucidate details of the structure-function relationships of PARP1. This research is not only important for unravelling the mechanisms underlying drug resistance, but it may improve future treatment plans for cancer patients. In this webinar, you will learn: - How to use genome-wide CRISPR screening for mutant discovery - How to create a highly diverse, sgRNA library from Twist Bioscience for targeted, subtle mutations - How knowledge of the structure-function relationships of PARP1 mutants can inform treatment of cancer patients with these drugs

Activation of immune cells is the all-important first step in mounting an immune response. Immune cell activation is a popular area of research because so much happens that is key to the downstream goal of fighting infection, cancer, and disease. There are many ways to measure immune cell activation, and they all have utility. Methods can be grouped into four main categories: Proliferation Assays, Cytokine Measurement, Surface Antigen Expression, and Cytotoxicity. In this webinar, we'll discuss specific assays in each of these categories, the joys and pitfalls of each assay, and recommendations on how to choose the best method. You will learn tips and strategies for successful assay development using the following methods: Proliferation: - 3H-Thymidine Uptake - Bromodeoxyuridine Uptake (BrdU) - ATP Luminescence - Fluorescent Dye Reduction (CFSE) Cytokine Measurement: - Multiplex vs. Single Cytokine - Choice of Cytokine (IFNg, TNFa, IL-6, IL-1?, etc.) - Kinetics of Cytokine Release Surface Antigen Expression: - CD69, CD25, PD-1, etc. - Combine with CFSE, Ki67 or BrdU - Kinetics are Important Cytotoxicity: - Two-Label Flow Cytometry - Calcein AM Dye Release - Luciferase Transduced Targets - Annexin V

While next generation sequencing enables researchers to unveil expression levels of the entire genome, qRT-PCR remains the gold standard for measuring transcript levels of individual genes for functional studies and for the purposes of publication. In this webinar, you will learn: • Low (1-5 genes) vs medium (~300 genes) throughput experimental design • Pros and cons of self-designed vs “off the shelf” assays • How to set up your wet lab experiments start to finish • Downloadable example step-by-step experiments with real data analysis and tutorial • Biological considerations (time series data, cell population frequency changes + more) • Examples of these techniques in publications • Common pitfalls and how to avoid them • Limitations of the technique • MIQE and publication standards Whether you are interested in a few genes or a few hundred, join Matthew Mule as he takes you through the necessary steps to validate expression levels of target genes using qRT-PCR with single gene assays and other medium-throughput platforms.

Join us in this webinar featuring Dr. Vicki Doronina as she takes you through vital components of assay design. In this webinar you will learn: How to choose between immortalized cells and primary cells for your assay How to avoid sources of bias in your cell-based assays How to use high throughput assays, so you can achieve greater reproducibility By now, you have heard about the reproducibility crisis—the inability of scientists to reproduce experimental results. This crisis spurred journals to institute new requirements for publication and funding agencies to introduce more stringent rigor and reproducibility criteria. In this webinar, Vicki will address an often overlooked source of experimental variability: state of your cell lines and their external conditions. The good news is that many of these factors can be addressed through assay design. She will show you how to move from poor reproducibility of so-called “artisan experiments” to the use of standard conditions for your cell lines and use of automatic systems for high throughput screens.

Join us in this webinar featuring Dr. Marlon Stoeckius as he explains how you can improve your single-cell RNA-sequencing (scRNA-seq) experiments. In this webinar, you will learn: - How you can run one scRNA-seq experiment with numerous protein markers in parallel - How you can increase your recovery of single cells (up to four times!) per experiment - How you can link phenotypes to transcriptomic profiles—with higher throughput methods! The last few years have seen the scale of single cell RNA-seq experiments increase exponentially, greatly enhancing our understanding of cell biology in development and disease. It is now feasible for researchers to characterize thousands of single cells in one experiment. However, important hallmarks of immune cell states are often not detected in scRNA-seq experiments. While lower throughput methods previously allowed researchers to link phenotypes or protein expression to transcriptomic profiles, the increase in scale of modern droplet-based methods resulted in a loss of such addressability. Here we describe two recently developed applications that utilize antibody-conjugated oligonucleotides to enhance existing scRNA-seq platforms. 1. CITE-seq, which allows measurement of a potentially unlimited number of protein markers in parallel to transcriptomes. 2. Cell Hashing, which enables sample multiplexing, robust multiplet detection and super-loading of scRNA-seq platforms, allowing confident recovery of 4 times as many single cells per experiment. Reagents for performing these assays, under the name TotalSeq™ are now available from BioLegend.

qPCR is one of the most specific and sensitive tools in molecular biology, allowing the quantification of target DNA molecules present at less than 1 in 106. Next Generation Sequencing (NGS) has similar potential. However, the presence of large amounts of non-target DNA in most clinical or environmental samples precludes easy and inexpensive analysis of rare events. In this webinar, you will learn: 1. The epigenetic differences between microbial and human/animal genomes 2. The use of restriction endonucleases to enrich for either pathogen genomes or the human genome from complex populations. 3. How the use of enrichment and concentration can improve qPCR sensitivity 4. The use of qPCR to validate enrichment from complex samples 5. The use of NGS to validate pathogen enrichment from complex samples Join Dr. Allyn Forsyth as he describes how successful NGS analysis of complex microbiome samples can lead to the development of non-invasive colorectal cancer screening, as well as monitoring of disease states within other cancers and Alzheimer's Disease.

Molecular interactions are key in cellular signalling. They are often ruled or rendered by the mobility of the involved molecules. We present different tools that are able to determine such mobility and potentially extract interaction dynamics. Specifically, the direct and non-invasive observation of the interactions in the living cell is often impeded by principle limitations of conventional far-field optical microscopes, for example with respect to limited spatio-temporal resolution. We depict how novel details of molecular membrane dynamics can be obtained by using advanced microscopy approaches such as the combination of super-resolution STED microscopy with fluorescence correlation spectroscopy (STED-FCS). We highlight how STED-FCS can reveal novel aspects of membrane bioactivity such as of the existence and function of potential lipid rafts, and how the new FALCON technology eases such measurements.

Elucidating meaningful, unbiased microbial community profiles from complex microbiome samples is challenging. In this webinar, you will learn: – the sources of bias throughout the microbiome analysis workflow – practical solutions for troubleshooting your techniques – new technologies to achieve the most representative and unbiased microbiome profiles Join Dr. Sven Reister as he guides you through a typical workflow for analysis of microbial community profiles of complex and low biomass microbiomes, and learn how to get the most complete, unbiased microbiome profiles from even your most challenging samples.