...plasmid to express the target antigen could be screened in a high-throughput manner. Cells were labeled...which was observed for the hybridoma mAb.  While screening by IF-ICC worked well for identification of the...was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas and, ...limited the number of clones that needed to be screened through functional assays. Following functional...

...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...Sendai virus doesn’t rely on promoters for transcriptional regulation, so it requires a different approach...in practice integration can occur. Okita et al describes a protocol for generating iPSCs by plasmid transfection...Methods RNA Delivery: For this approach, in vitro transcribed RNAs are serially transfected using a cationic...transduction, such as the polyarginine peptide described in Zhou et al and Kim et al. This is considered...

...read more content related to CRISPR technology, subscribe to our CRISPR blog content.   In this post we ...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...deletions and introduce epitope tags and RNA transcription start sites. The lab further demonstrated that...comprises an active Cas9 fused to the reverse transcriptase (RT), which induces two double-strand breaks...

...indicators. Tian developed a video-based high-throughput screen for directed evolution of biosensors based on their... to engineer many types of biosensors.   This screening method allowed Tian to optimize a red‑shifted ... the improved voltage indicator coming from the screen to understand mouse cortical and hippocampal neural...neural microcircuits. “In the longer term, this screening platform may help us to create high-performance...

...may not result in any cells, or all the colonies screened might not contain the correct plasmid. There’s...pick the big colony and you are safe! Colony screening Pick a few colonies (5-10) and grow them in a ... you've identified potential clones after the screening, sequence the plasmid. Many vectors contain sequences...can use the chromatogram to spot whether any discrepancies between your sequencing result and the expected...

..., the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit of this...the organisms present while sequencing of all transcripts gives clues into what the microbes are doing....delivery into recipients by conjugation. The labs screened for the host-range of the plasmids and suitability...

...ligands NRs represent the largest family of transcription factors found in metazoans. The superfamily ...profiling datasets in to our data mining tool, Transcriptomine. We are currently nearing the completion of...transient transfection platform for assaying transcriptional activity, for example, was a key development...coregulators: Judges, juries and executioners of transcriptional regulation. Molecular Cell 27, 691-700. (PubMed...

...includes overview information, key dates, funding description, research plan, award information, eligibility...aspects are understood. While reading the funding description, pay attention to the keywords used and think...grant applications restrict the space available to describe the proposed work. Thus, every single sentence...project are clearly stated and align with the description of the RFA and funding institution. Emphasizing...

...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...) complementary to the genome and a reverse transcriptase (RT) template including the desired edit. ...., 2019). Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...

... by T7 promoters to allow for bidirectional transcription to create dsRNA. Students can produce DNA templates...A recent publication [1] from the Sánchez lab describes the plasmids and protocols required for both WISH...the temporal and spatial localization of mRNA transcripts in tissue. This is useful for labeling specific... which are available through the deposit) are described in the Sánchez lab publication [1] so that the...

...loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a colorimetric readout that can... 45 minutes. The team deposited the reverse transcriptase, protease, and Bst polymerase constructs from...toolkit include ones for expressing MMLV reverse transcriptase and Taq polymerase that can be used in a one-step...Hsu PD (2021) Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating...

...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc 12:828–863 . https:/...ask the depositing laboratory to review the discrepancies. If the depositing laboratory confirms that ... use a modified version of the Python program described by Joung et al. 2017, which we hope to make available...

... protein to date By Justin Ng High-throughput screening methods, such as fluorescence-activated cell sorting...s lab developed a new form of FACS-based cell screening, Single-cell Phenotypic Observation and Tagging...plasmids here Reduce the scale of your CRISPR screens by using this lentiviral CRISPR human genome-wide...guides per construct and allows a reduced scale in screens without any reduction in performance compared to...

...for viral titration consider using a reverse transcriptase mutant in a separate control reaction. This ...results. A packing vector deficient in reverse transcriptase will produce low levels of virus and can be ...into a target cell’s genome. This approach can discriminate multiple integration events, reducing the possibility... How Lentivirus is Used in CRISPR Genome-Wide Screening Learn How Lentivirus Has Been Used for Lineage...

...Maria were encouraged by previous reports that described magnetic responses of cryptochrome proteins or... and the team set up a directed evolution and screening experiment with three of the proteins that had...several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet variants were showing no... suggest she deprioritize the project, Maria’s screens of AsLOV2 variants showed some significant gains...

...how much data is necessary before submitting a manuscript. In fact, how many of you have negotiated over...over the order of authorship for a manuscript? And that’s only in your scientific life! I’m certain that...mate would have time to make comments on your manuscript. If you give her this slot, she’ll have extra...her deadline) to help you. Her feedback on your manuscript is more important to you than collecting your...

...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...obtained if any single component was omitted. Screening for successful Golden Gate Assembly All Golden...to faster assembly workflows that require less screening to confirm the accuracy of assembly. The fidelity... assemblies per plate using outgrowth volumes described in Figure 3, with calculated yields from entire...

...up, most will eventually grow within a week. We screen the wells in groups as they become confluent, taking...important to have a functional assay in place to screen your clones. Depending on the protein, we have ...SH-SY5Y cells, as these cells did not seem to form discrete colonies but constantly migrated away from each...either initially, or using secondary biological screens. For example, for cancer researchers, if growth...

...proteins involved, with the goal of being able to discriminately and effectively eliminate cancerous cells from...lines. Further, for each plasmid in the kit, the transcript most commonly found across all TCGA tumor samples...he or she can use to study it (as seen in the screenshot below). Currently, the page includes interactive... Tackling Cancers' Drug Resistance with a New Screening Kit Learn How the Clon Tracer Library Can Be Used...

...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label ...world examples of these situations in cells are described below (See Figure 1).   Figure 1: Each... not necessarily monomeric, even if they were described as monomeric in a publication. The gold standard... [2]. Some affinity assays used in the papers describing the FPs above including molecular sizing columns...