...number of cycles.  Choosing a method  As with all experiments, a number of factors will determine which approach...Resources needed Most molecular biology labs and departments will have qPCR machines available for use. ddPCR...Chung WK. Quantitative analysis of copy number variants based on real-time LightCycler PCR. Curr Protoc... The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative...

... it can amplify DNA using only a short list of reagents and several heating and cooling steps. PCR relies...strand of DNA to add nucleotides to.  Using these reagents and a series of heating (denaturing) and cooling...cycle. However, it is common for dNTPs and other reagents to run low during the final cycles which would...of different molecular biology techniques and experiments. End point PCR’s goal is to amplify a specific...

...post-transfection to help remove toxic transfection reagents and keep the cells happy. Harvesting virus Once...widely used method due to the accessibility of instruments in standard labs. When determining titers by ...Adeno-Associated Viral Vectors by Oxidant-Based Disinfectants. Human Gene Therapy, 32(13–14), Article 13–14...sensitivity sufficient to detect single infectious events. Human Gene Therapy, 15(7), Article 7. https://...

...the degron domain is terminal. Between the tag elements (protease and degron) and the protein of interest...other systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir...reviewed here. The AID system relies on three components. First, the protein of interest must be AID-tagged...SMASh). If you don’t want to deal with additional components beyond the minimum required for a degron system...

...produced in multiple different cell types, including plants, mammals, and insects. In this post, we will compare...producing virus within 24 to 48 hours. Harvesting timepoints will depend on your specific virus, so this is...methods like ultracentrifugation, while lentivirus supernatants are centrifuged and filtered to eliminate any... important to you — virus type, cost, time, or reagents and equipment. Hopefully this post has provided...

... corrective amounts of protein for any such disease. This technology therefore represents a potential ...In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516:423...doi.org/10.1038/srep05105 Stephens CJ, Lauron EJ, Kashentseva E, Lu ZH, Yokoyama WM, Curiel DT (2019) Long-...

...particularly related to the neurosciences.  These developments in microscopy coupled with techniques that make...view of a cleared rat pons, medulla and the first segments of the spinal cord showing the cortico spinal ...spinal tract, CST, spinal cord and cranial nerve afferents labelled with a AAV8 expressing eGFP under the human...anterior, P= posterior, D= dorsal, V= ventral Developments in light sheet fluorescence microscopy Modern...

...is the first of what I, scientists, doctors and patients hope are many more clinical successes for gene...the human condition. Luck brought all these key elements together at the right time and place. But it is...limitation, with limited success so far. Luckily for patients, this gene can be targeted to RPE cells using ...improve the technology: engineering of new capsid variants, designing expression cassettes to regulate and...

...require a standard or a reference, meaning saving on reagents (and time)! AAV titering using droplet digital...emulsion technology. Each droplet contains the ingredients for amplification of the target DNA. This partitioning...prepare your sample dilutions. Aliquot all of your reagents into single-use tubes and grab a fresh one for...digital PCR is becoming a popular option for PCR experiments.  References Furuta-Hanawa, Birei, Teruhide...

...give to post-docs and graduate students as they complete their appointments in your lab?  Fujii: These days... makes it much easier to knock-in LexA binding elements into the target region. However, it struck me ...Throughout your career, you have held research appointments in the US (New York University School of Medicine...

...questions and comments go up early in the day, and the moderators were on top of any comments that were at...Anything”) on r/Science. You can see some of Joanne’s comments on the AMA process below, but we also wanted to...Protocols: Using Apps to Save Time & Track Your Experiments Additional Resources See How We're Sharing ...

...annealing can be affected by the presence of protein contaminants, which is a factor in AAV titration since the...repeating the exact same PCR twice can give titer measurements that vary up to two-fold. How can we get more...low levels of background protein and chemical components, ddPCR may not be an improvement over qPCR (Taylor...). What do Addgene AAV titers mean for your experiments? Because (physical) AAV titers are approximations...

...significant, negative effects in RNA targeting experiments. If Cas7-11 lacked this toxicity, it would represent...forward for CRISPR-based RNA editing - and further experiments strongly supported their initial findings.  The... approach to gene replacement therapy for most patients. But how does one go from “cut and paste” to “...Programmable Addition via Site-specific Targeting Elements (PASTE). Finding the right integrase for this ...

...exercise: Consider the issue as if there were no restraints on your problem-solving resources. What would...which people were asked to create a device to help students during an interview. When they were instructed... ideas heard.  So how can we assure that all participants feel comfortable sharing their ideas? And how...have plans in place to be able to continue our experiments. Thinking about these plans before you’ve run...

...specific QC experiments performed varies for each viral lot. To learn which specific QC experiments were performed...ddPCR) using primers and probes targeting the ITR elements and an internal control of known titer (protocol...viral genome and the absence of unexpected DNA contaminants. The data is also used to determine recombination...

...materials, we noticed a large percentage of these shipments arrived in India at room temperature. Partnering...distributor for that country. In China, direct shipments weren’t able to clear import control. Partnering...any conference is scientists, from techs to grad students to professors, stopping by to say, “Everything...— in their classroom, in their lab, in their experiments, to enable virtual learning during the pandemic...

...biorepositories or have been directly isolated from patients. In these cases, a test known as Short Tandem ... the lower the passage number the better for experiments. This is to ensure the cells are as close to ...original Cell line you purchased, keeping all experiments carried out on low passage cells and this means...and we can progress faster to desired clinical endpoints.   Nick Amiss is an ATCC Outreach Scientist at...

... type of memory which includes autobiographical events (see Jin & Maren, 2015). Directional flow of information... and memory retrieval. In neuronal tracing experiments, brain tissue is injected with compounds that...injection or iontophoresis (applying small electric currents via inserted electrodes) may be applied, but these...proteins VP1, VP2, and VP3. To date several hundred variants, or serotypes, have been found naturally or have...like these may be a starting point for future developments in the field of AAV vectors. Conclusion An ideal...

...smaller fragments of the transgene have been seen with the hybrid method. While these fragments are likely...co-infected with both part A and part B AAVs, the fragments reassemble and produce the full-length gene. Below... donors for two consecutive CRISPR-mediated HDR events which results in the integration of a larger donor...

...have considered myself neutral in any of their disagreements. In fact, conflict resolution isn’t just about...also be good. In science, a reviewer’s critical comments on your manuscript is a form of (hopefully constructive...constructive) conflict. As you perform the experiments to address a reviewer’s concerns, you are making... around missed tasks, stalled projects, and disagreements over prioritization. The source of those conflicts...