...pCINeo-hFX-HA FXN HA, AU1 CMV Friedreich ataxia Michael Ristow 14977 pCINeo-hFX(I154F)-HA FXN HA, AU1 CMV...pBlue-hFX-HA FXN HA, AU1 T7 Friedreich ataxia Michael Ristow 14988 pBlue-hFX(I154F)-HA FXN HA, AU1 T7 Friedreich...pBlue-hFX(T133A)-HA FXN HA, AU1 T7 Friedreich ataxia Michael Ristow 14990 pBlue-hFX(T133E)-HA FXN HA, AU1 T7 Friedreich...-hFX-HA FXN HA, HIS, AU1 T7 Friedreich ataxia Michael Ristow 14992 pET28(a)-hFX(I154F)-HA FXN HA, HIS,...AA88)-HA FXN HA, HIS, AU1 T7 Friedreich ataxia Michael Ristow 15403 pBlue-hFX(I154F)(AA88)-HA FXN HA, HIS...pBlue-hFX-HA(AscI) FXN HA, AU1 T7 Friedreich ataxia Michael Ristow 15405 pBlue-hFX-HA(NcoI/EcoRI) FXN HA, AU1...Camilli 22563 Flag-HA-UCHL1 UCHL1 HA, FLAG Parkinson's Wade Harper 22594 Flag-HA-OTUD4 OTUD4 HA, FLAG CMV Gordon...

.... Cas9 nickase (Cas9n), a D10A mutant of SpCas9, has one active nuclease domain and one inactivated one... available. SaCas9 (from Staphylococcus aureus ) has a coding sequence ~1 kb shorter than SpCas9 while... SpCas9. FrCas9 (from Faecalibaculum rodentium ) has increased fidelity, and recognizes NNTA, a PAM sequence... create the adenine base editor. The TadA domain has also been further evolved to catalyze cytidine deamination...editing requires more optimization and typically has lower efficiency (< 50%) than base editing (> 90%...modifications to eliminate target gene function. It has also been used extensively to screen for novel genes... those for yeast or drosophila . Although CRISPR has been less widely used in bacteria due to technical...

...purchased at Addgene. Each transcription factor isoform has a unique 24 bp barcode that simplifies identification...ID PI Description MORF library 137000, 192821, 1000000218 Zhang A collection of human transcription factor...

... your gene of interest once its appropriate cDNA has been fused to the MS2 binding sites within this plasmid...expression only at a specific time. This collection has a variety of plasmids whose expression levels have...in response to green light. BIOFAB plasmid set 1000000037 N/A N/A Drew Endy This kit contains plasmids ...

...protein–protein interactions. The Roux lab has deposited HA tagged BioID2 for N-terminal fusions and Myc...produces point mutations at a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate... activation of these receptors. Bryan Roth's lab has a newly available PRESTO-TANGO kit which allows researchers...the NCI RAS Initiative, led by Dominic Esposito, has recently released a collection of 360 Ras pathway...24395366 Oxford Drosophila CRISPR library Addgene has had the privilege of distributing several human and...this 3-day goal is is now attainable. The Jacks lab has recently introduced a new assembly cloning platform...Addgene to help expand the collection! The Jacks lab has made several GMAP-compatible plasmids available to...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller...96-well plate, seed one of the corner wells with ~1000 cells. This will help you focus the microscope later... incubating the cells. Pro-Tip Use the well with 1000 cells to focus the microscope, then you can easily...starting this procedure, be sure that the cell pool has been sufficiently selected with the appropriate antibiotic... antibiotic so that every cell in the culture has, in theory, been transduced. There are various schools...regularly for transgene expression. This protocol has been optimized for adherent cells but can be modified...

...mL conical collection tube. Centrifuge column at 1000 x g for 2 min to remove storage solution. Place ...container. Add 5 mL PBS to the column. Centrifuge at 1000 x g for 2 min to remove PBS. Repeat Steps 30–31 ...resin bed to fully absorb the sample. Centrifuge at 1000 x g for 2 min to collect the sample. Discard column...). Pro-Tip Add protease inhibitors to 1X if this has not already been done. We typically start with about... NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. If the sample ... NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. Gently transfer... Adjust pH to 7.0 Autoclave or filter sterilize 1000X protease inhibitor cocktail 25 mg leupeptin 50 mg...

...ensure that it has not drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Hemocytometer or cell ...system, Nalgene, 565-0010 (or for larger volumes 1000 mL 0.45 µm Rapid-Flow PES Filtration Unit, Nalgene... deionized water and adjust to a final volume of 1000 mL. Stir at RT until fully dissolved. Pro-Tip This...clogging during harvesting. The addition of sorbitol has been shown to increase viral titers by up to 1.8-...such as sorbitol and sodium bicarbonate, sorbitol has been found to increase viral titers by up to 1.8-...

...plasmid #), and other reagents. F1000Research, among many other journals, has joined a pilot project from ... up their own minds on whether the data analysis has been of good quality and whether they agree with ...Maaike Pols, PhD, a Developmental Editor at F1000Research.  With many changes taking place in the world...results more valuable. Some journals, including F1000Research, also ask for the raw data behind the figures...several rounds of revision before acceptance. F1000Research uses a novel form of peer review that occurs... Maaike Pols is a Developmental Editor for F1000Research, responsible for assessing the scientific content...

... a raise in salary, particularly given that this has precipitated increases to the NIH recommended pay...both of these changes share a common thread that has the potential to profoundly shift the academic science...rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy of academic science...sector throughout their careers (McDowell et al, F1000Research, 2014; Dolan, F100Research, 2016). These events...Research: a perspective from junior scientists."F1000Research 3 (2014). Pubmed PMID: 25653845. Pubmed Central...from the Future of Research Chicago Symposium F1000Research, 2016. Kyle T. Dolan, Joseph F. Pierre and Erin...

...ensure that it has not drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid...

...Cas9, but the ever-expanding repertoire of CRISPR has brought many other Cas effectors to light. One lesser-known...effector, and type IV with an unknown effector. Type I has subtypes A through G, with different compositions... Thanks to this complex, developing Cas3 tools has been challenging. It can be difficult to ensure comparable... job — Cas see DNA, Cas chop DNA. Cas3, however, has two jobs, one as a nuclease and one as a helicase...easier to work with than Cas3. Future targets Cas3 has the potential to be incredibly useful in therapeutics...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 O’Brien, R. E., Bravo, J...

...expensive, it may be helpful to find an antibody that has already been validated for flow cytometry through...Characterization through Open Science) initiative has now started producing flow cytometry characterization...overexpression. Additionally, if the cell line used has endogenous levels of expression, this can make the...TIM-1 (Single pass type-1 membrane protein), which has been designated CD365 in the most recent HLDA workshop...as part of the YCharOS open science ecosystem. It has received in-kind support to produce this data from...and flow cytometry. F1000Research, 13, 817. https://doi.org/10.12688/f1000research.154034.1 Laflamme, ...synaptotagmin 1 page. The data produced is available on the F1000 gateway and Zenodo community.  Knockdown approach...

...filter, VWR 431229 0.22 µm PES filtering system, 1000 mL, VWR 431098 0.45 µm PES filtering system, 500... aliquots and store at -20 °C until use. BCD TFX 1000 mL BalanCD HEK293 Media 10 mL 10% Pluronic-F68 40...final volume of 1 L and recheck pH to ensure that it has not drifted. Filter the solution through a 0.22 µm...incubate for 1 h at room temperature. After the BCD TFX has warmed in the bead bath, transfer the tubes to the...suggest preparing fresh solutions after one month. 1000X protease inhibitor cocktail 25 mg Leupeptin 50 mg...

...excision site after cleavage by the transposase. It has a cargo capacity >100 kB, although integration efficiency...efficiency decreases with cargo size. Sleeping Beauty has a close-to-random integration profile in mammalian...hypersensitivity sites. Like Sleeping Beauty, piggyBac has an integration efficiency in human cells similar ...1996). Unlike Sleeping Beauty and piggyBac, Tol2 has a weak consensus sequence for its preferred target...hypersensitivity sites. Tol2 is only active in vertebrates and has lower efficiency of integration in human cells than... is a truncated version of the original Tol2 and has ~3-fold increase in transposition activity  Tol2...encodes a Type I-F CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA (crRNA), 2...

...amplified and none are lost. Each Addgene library has its own depositor transformation and amplification...product for next-generation sequencing Once the DNA has been extracted, the representation of the library... success of your amplification. I hope this post has helped clarify some of the important considerations...you want to have 100x-1000x coverage of your library: in other words, 100-1000x more reads than number...

...HDR, NHEJ is active throughout the cell cycle and has a higher capacity for repair, as there is no requirement...ends interfere with that step. Accordingly, NHEJ has a vast toolbox of processing factors, including polymerases...Potential. PLoS Genet 5:e1000683 . https://doi.org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P...

...ensure that it has not drifted. Filter the solution through a 0.22 µm membrane. Aliquot 500–1000 µL into sterile...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 20–200 µL multichannel...Microcentrifuge tubes, Neptune 3745.X Pipette tips, 1000 µL, VWR 76322-154 Pipette tips, 10 µL, VWR 76322...periodically to determine when the desired color change has occurred. The color will change from yellow to blue...experimental conditions. When the desired color change has occurred, gently remove the plate seal. Using a multichannel...

...can’t it’s an easy search. Writing this tutorial has shown me that I’m still in the dark ages with respect... organization issues – F1000 just announced the launch of their F1000Workspace.  Give your favorite searching...Maaike Pols, PhD, a Developmental Editor at F1000Research  ...