... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...,000 TU/mL For a more accurate titer, take the average of multiple dilutions. Sample Data Figure 1: A549...

...look at the side of an objective, you will see several pieces of information, but the most important (...your observations. Some microscopes have digital cameras attached to them that allow you to capture images...

...gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified... gel, make 10% more volume than needed because several microliters can be lost in pipetting. For example...

...Existing therapies and effects on immune system Conventional cancer therapies include radiation therapy (owing... Types of immunotherapeutics In lieu of the cancer-immune system interactions, therapeutics have been ...Curie), chemotherapy, and surgical removal of the tumor. However, these conventional therapies produce...cell-surface tolerance-producing proteins are one of the most commonly used immunotherapeutic agents. Currently...contestants for cell-based immunotherapy. Using activated DCs ex vivo to generate cytotoxic effector T cells...beneficiaries. Collaborative therapy For the most efficient outcomes, collaborative therapies must be explored. ... multiple immunotherapy targets can be utilized at the same time. A combination of tolerance marker blockade...

...combination of the promoter (RNA polymerase binding site) and operators (response elements). Promoters ...      The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria...     Description Expression Additional considerations CMV General expression mRNA Strong mammalian expression...PGK1 (human or mouse) General expression mRNA Mammalian promoter from phosphoglycerate kinase gene.  Constitutive...promoter with low basal activity and several tetracycline operators. Transcription can be turned on or ...promoter. GDS General expression mRNA  Strong yeast expression promoter from glyceraldehyde 3-phosphage ...Description Expression Additional considerations T7 in vitro transcription/ general expression Promoter from ...

...under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression of the...allows genome editing and mutant library generation in several biotechnologically and clinically relevant...proteins. Overall BioID2 improves the efficiency of screening for protein–protein interactions. The Roux...on protein-protein interactions and therefore identifying and studying interaction networks is important...Protein A-Renilla luciferase and the “prey” proteins are tagged with Firefly luciferase. The bait and prey...A tag. The luciferase activities can then be measured and normalized to assess interactions. Higher relative...firefly luciferase activity in the Co-IP compared of a control Co-IP indicates stronger interaction. The ...

...Current CRISPR Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer...delivery and editing strategies for CRISPR therapies. Systemic therapies involve CRISPR-Cas delivery directly...next few months. Several other companies have followed suite, with Beam Therapeutics initiating a base... initiated a CRISPR therapy for hATTR with the first-ever systemic CRISPR therapy. They engineered a CRISPR-Cas...this therapy is affecting hATTR patient outcomes. Bacterial infection and HIV Most CRISPR therapies have...part of a cocktail therapy for urinary tract infections. The Cas3 enzymes target several strains of E. coli...sponsored by Excision Biotherapeutics. Additional ongoing trials The CRISPR therapies don’t stop with the...

...spatial and hierarchical chromatin interaction networks. Think of these chromatin interactions networks ...pictures” generated with current 3C methods provide useful information about chromatin interactions, they ...could detect multiple chromatin interactions at once. Chromatin interactions at five sites in the well-characterized...chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously...Determining the spatial and hierarchical organization of chromatin interaction networks  A second application...enhancers are interacting with which promoters, what part of a gene enhancers like to interact with, and ...that forms chromosomes. These long range DNA interactions are often detected with chromosome conformation...

... that the ligation regenerates the original restriction sites for further iterative cloning steps and ...and voltage sensitivity. This hierarchical screen led to the generation of two enhanced voltage indicators...Instead, coelenterazine is supplied to the culture media as a chemical substrate for Renilla luciferase. This...alternative to the widely used luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids... issues with fluorescent pH sensors, a set of luciferase-fluorophore pH fusion reporters, pRSETb-pHlash...insensitivity to other ions In BRET, A mutant Renilla luciferase, catalyzes the oxidation of its substrate, luciferin...control of signalling pathways. These LIDs are generalizable, versatile and powerful tools which allow you...

...Second-generation vectors are likely sufficient for most experiments; however, the 3rd-generation packaging... 2nd vs. 3rd Generation Lentiviral Plasmids Q3: How can you tell 2nd- and 3rd-generation transfer vectors...considered safer. Second-generation transfer plasmids MUST be packaged with a 2nd-generation system because the... Third-generation plasmids can be packaged with either system. Please note that the generation used to...limiting the interaction between viral LTR enhancers and the transgene; such interactions can alter expression...production (3 or 4). Third-generation vectors contain gag, pol, and rev. Second-generation contains those three...and the plasmid coding for an envelope. Three generations of lentiviral packaging systems have been developed...

...protein-protein interactions, RNA-protein interactions, and even more recently cell-cell interactions. If you’... binary protein-protein interaction as well as identify additional interacting proteins in one simple ...RNA-protein interaction detection So far, we’ve covered ways to study protein-protein interactions, but scientists...RNA-protein interactions that can regulate multiple biological functions. RapID (RNA-protein interaction detection... mammalian cells (Milo, 2013). The number of interactions between these proteins is hard to imagine yet...yet alone study. To elucidate these interactions scientists have long used antibody based affinity purification...proximity-based labeling to better capture these interactions in a vast array of biological contexts.  Proximity...

...Apart from fibroblasts, human keratinocytes from hair pluck, peripheral blood cells, and renal epithelial...Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive...Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol,...Mukherjee. The ultimate goal in the field of regenerative medicine is to replace lost or damaged cells...cell is converted to a different cell type for regeneration and repair and situations where one process ...reverts back to a pluripotent state, which then proliferates and is redifferentiated to a different cell ...stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate to another cell type of choice...

...molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions. The...case a reporter gene (e.g. luciferase or GFP), regardless of whether it interacts with a transcriptional ... the split-luciferase system, intein-mediated splicing reconstitutes functional luciferase only when fusions... fusions of the two halves of the luciferase protein interact and enable intein splicing (5). Screening...Umezawa, Y.l. Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells...create a new system for screening protein-protein interactions. In later years, improvements to yeast transformation...the classic two hybrid system (also called the interaction trap), genes encoding proteins of interest(POI...

...her shirt. She rolled several long takes, and we cut it up in post.  Test: Overall, it worked. We pulled...someone to be comfortable on camera while thinking about things like lighting, camera angle, and shot composition...and wearing their mask. But while the camera is here, the operator is in a different room.   Design... best possible videos for free. We’re a small operation, so sometimes it can be a big challenge. But ask...had a couple options. The first was to put me on camera, filming myself. While easiest from a production...protocol and can guide viewers through. I know cameras, but we needed someone else. Design: Jennifer thankfully... the office, we use a Black Magic Pocket Cinema Camera 4K. It’s wonderful and simple to use, but when ...

... gel, make 10% more volume than needed because several microliters can be lost in pipetting. For example...

...treatments, such as surgery, radiation, and chemotherapy, immunotherapy is coming to the fore as a cutting edge...performs a heated chemotherapy wash throughout the abdominal cavity. This targeted chemotherapy approach has...susceptible to up and coming immunotherapies. The standard definition of immunotherapy is the use of drugs (or...research opportunities are being provided to immunotherapy than ever before. Although different types of...great promise in up and coming treatments like immunotherapy. Mesothelioma Mesothelioma is a terrible disease...first mesothelioma symptoms in their body. The average latency period (time from first asbestos exposure... certain other naturally occurring asbestiform minerals such as erionite, are the only scientifically ...

...best). The moderator takes control It is the moderator's job to prevent anyone from derailing the show....Choose a moderator with a backbone. It is nice to have a subject area expert, but the moderator is not ...LinkedIn profiles of all panelists to the moderator. The moderator and organizers should develop a list of... the interaction is not interesting to the audience. It is a bit of an art, but active moderation gets...effortless to the audience, but the organizers and moderator should have a well-planned approach to making ... of 4-6 pre-event interview questions. The moderator (possibly with an organizer) should set up a 30 minute...to schedule. In the pre-event interviews, the moderator should be taking good notes and discovering interesting...

...you need a high-fidelity polymerase. There are many high fidelty polymerases on the market; you need one...a DNA polymerase that produces blunt-ended PCR products (e.g. Phusion, Pfu, and Vent polymerases). Note...Note that certain polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary...site in the coding region (preferably near the 5’-end) and that also generates 4 bp overhangs. You can then... then use the Klenow fragment of DNA polymerase I to generate blunt ends from these overhangs and ligate... analyzing the role of renaturation temperature in the polymerase chain reaction." Analytical Biochemistry...this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome...

...far, with safety increasing in each generation. The 1st generation of lentiviral vectors consisted of ...third-generation lentiviral vector system offers the best safety profile in terms of RCL generation because...2nd and the 3rd generation of vectors have been shown. However, with the 3rd generation you get lower viral...-create a functional virus. Indeed, by testing several vectors and packaging cell line combinations, Miller...were produced in high-titer retroviral stocks generated by the transfer of a retrovirus into the packaging...vectors Although retroviruses are still in use, considerable efforts have been devoted to develop efficient...infect non-dividing cells. Thus three different generations of lentiviral vectors have been established so...

... comes from using Taq polymerase for the amplification step since Taq polymerase leaves a single deoxyadenosine...in the pair comes from a topoisomerase I-linearized backbone. DNA topoisomerase I (depicted as a green ...covalently linked to topoisomerase. Vectors in these kits also often have the topoisomerase site inserted into...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes...technique specifically uses Vaccinia virus-isolated topoisomerase I as this enzyme recognizes the DNA sequence...cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different...