... Commercial Plasmid Prep Kit Amount 1000 Lanes 1000 Lanes 1000 Lanes Cost $200-$500  <$10 $20-30 ...convenient, commercial ladders cost between $200-$500 for 1000 lanes worth of ladder. The Penn State ladders are...plasmid prep protocol, it cost less than $10 to make 1000 lanes worth of the 100 bp or the 1 kb ladder. Using...Your Agarose Gel Other Resources Read about Gel Stains on the BitesizeBio Blog ...

...fight in the Napoleonic Wars and against something even more sinister. 1000 pages long. Incredibly imaginative...larger epic of fantasy novels. Set in a world where 1000 years ago the hero lost and the Dark Lord rules ...founder, Eric Perkins, is Addgene’s Director of Science Product Management and explains the origins of ABC: ... favorite books - both scientific and not - to inspire you to read a little more this October. Science..., examining relationships between microbes and insects, mammals, and plants, among others. I particularly...and with the events of the times."     - Aliyah Weinstein, Marketing and Communications Manager Addgene's... Aliyah Weinstein...

...unique sgRNAs along with 1000 non-targeting controls. This backbone contains SpCas9. SpCas9 and 76,441...genes and containing 76,441 unique sgRNAs along with 1000 non-targeting controls. This backbone does not contain...unique sgRNAs targeting 19,114 human genes along with 1000 non-targeting controls Puromycin Doench , Root 73179...unique sgRNAs targeting 19,114 human genes along with 1000 non-targeting controls Puromycin Doench , Root Human... unique sgRNAs targeting 19,674 genes along with 1000 non-targeting controls. This backbone does not contain...unique sgRNAs targeting 19,674 mouse genes along with 1000 non-targeting controls Puromycin Doench , Root Cas9...our repository that express fluorescent proteins. ID Name Insert Antibiotic Resistance Purpose PI 17446...

... but a select few are particularly special. Over 1000 of our 60,000+ deposited plasmids have been requested...Said Blue Flame awardee Sophien Kamoun of the Sainsbury Laboratory in Norwich, UK: “We’re very pleased...

...barcodes. Rather than continuing to process 100s to 1000s of samples individually, plexWell™ reduces every...help unlock the potential of modern sequencing instruments by enhancing the efficiency and simplicity of...completely unnecessary for sequencing multi-kb DNA inserts and larger targets. The NGS readout also reveals...

...pieces of roughly equal size, typically between 200-1000 bp. This is to ensure that your downstream analysis... antibody on crosslinked proteins. In the case of histone modifying proteins, which bind quite strongly...that ChIP uses native proteins (so antibodies validated only for denatured proteins may not be a good choice...another. For instance, a sample may need more or less cross-linking to firmly attach the proteins to the DNA...is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works by using...exploring these interactions, and can give valuable insight into DNA expression and protein interactions, including...the downstream analysis process. And ChIP itself begins at...  Crosslinking Once you have your sample, ...

...proximity ligation." Nature methods 3.12 (2006): 995-1000. PubMed PMID: 17072308.  4. Dieck, Tom, et al. "...incorporated into newly synthesized proteins instead of methionine. The labeled proteins can be detected using click-chemistry...turnover of individual proteins in situ.  For the visualization of specific proteins in situ immunocytochemistry...audiences. Right now, she is a science instructor at The Innovation Institute in Boston. When Eugenia is not ...protein is decreased in Alzheimer’s disease patient’s brains. However, it is not known why or how this happens...degradation. These processes control the abundance of proteins in cells. Homeostatic regulation of protein abundance...type. However, we cannot distinguish new from old proteins within that pool. Therefore, it is not possible...

...and very cool 8,768,560 hours – or almost exactly 1000 calendar years!   *Dammit, man, I’m a scientist... to make a plasmid. Doench’s lab in the Broad Institute has created hundreds of thousands of plasmids,...Table 1.   Of course, this is for the simple insertion of a PCR product into a vector. The time it takes...your experiment. Make your work “high-throughput” Instead of making one plasmid at a time, try constructing...

...particles. Studies have found that values can be 10-1000 fold higher than for functional assays depending...The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected...

...coefficient and quantum yield of the protein, divided by 1000. In many cases the brightness is compared to that... photoconvertible proteins are PA-GFP, Dendra2, and the mEOS proteins. Some proteins are reversibly switchable...you really know how fluorescent proteins glow?   Fluorescent Proteins (FPs) were first discovered over...categories of fluorescent proteins Photoactivatable / Photoconvertible: These proteins can switch their color... less).  For LSS proteins, the Stokes shift is ≥ 100nm. Specifically, these proteins are excited by UV...when labeling proteins that interact. Indeed, FRET is often used to determine if two proteins interact. ...wavelength than the exciting wavelength. Thus, FPs are proteins with this unique capacity. Many of these FPs are...

...Beroukhim and Boehm labs pulled data from more than 1000 CRISPR screens in DepMap to see if they could identify... for instance, a recent preprint released by the Boehm and Beroukhim labs at the Broad Institute of MIT...MIT and Harvard, Dana-Farber Cancer Institute, and Koch Institute, where an exploration into the effects...introduced an ancestry bias into their experiments instead of a true correlation.   It turned out there was...

... (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of their target...Strong insect promoter from baculovirus Constitutive  Commonly used in expression systems for insect cells...to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which is necessary...responsible for initiating the transcription of your insert into RNA. In practice, the term "promoter" describes...regulatory factors: enhancers, boundary elements, insulators, and silencers; however, some "leakiness" of ...Strong hybrid mammalian promoter Constitutive  Contains CMV enhancer, chicken beta actin promoter, and...with Tetracyline or its derivatives.  Typically contains a minimal promoter with low basal activity and...

...used to calculate the viral titer: GC/mL = {[(R*C)(1000/V)]*D} R = Reaction volume C = Copies/uL V = Volume...using water-oil emulsion technology. Each droplet contains the ingredients for amplification of the target...experiment. The process to titrate AAV by ddPCR begins with diluting the virus. It is important to note...containing approximately 20,000 droplets. Each droplet contains the material required for a mini amplification...

...of template should have homology arms between 500-1000 bps. HDR efficiency with circular plasmid templates... generally preferred for large insertions such as fluorescent proteins or selection cassettes. This class...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Quadros RM, et...Stroik January 24, 2023. DNA damage drives genome instability and contributes to cancer, premature aging, and...sticky or blunt ends and often introduces small insertions and deletions along the way. HDR uses homologous...overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for ...template to create mutations HDR donors can contain insertable sequences or specific mutations, along with homologous...

...includes SpCas9, and shortern orthologs approximately 1000 amino acids in size. From the pool of shorter orthologs...split protein like Cas9 is to use split inteins. Split inteins are a pair of naturally occuring polypeptides...toxicity. More AAV-based CRISPR systems Smaller Cas proteins SaCas9 isn't the only CRISPR enzyme that’s small...advantage that it can be turned off by anti-CRISPR proteins. The Sontheimer lab used NmeCas9 with AAV to edit...disease-causing loci in mouse liver.  Two other smaller Cas proteins include Cas12b and CasX. While there are no papers...papers yet published that deliver these Cas proteins with AAV, at just 1,108 and 986 amino acids, respectively...polypeptides that, when at the ends of two proteins, mediates protein trans-splicing, similar to an intron...

...Low 572/ 610 FP595 Tetramer Green Dark/ Red 70 - 1000 Medium 590/ 600   Choosing a photoactivatable...FP-chimeric proteins. Photoactivatable fluorescent proteins (PA-FPs) are fluorescent proteins that display...Fluorescent proteins (FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate...Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral (protonated) and...irreversible PA-FPs are the photoconvertible fluorescent proteins (PC-FPs). PC-FPs can convert from one fluorescent...Selected Irreversible Photoactivatable Fluorescent Proteins Protein Oligomeric State Activating Light ...Selected Reversible Photoactivatable Fluorescent Proteins Protein Oligomeric State Activating Light ...

...design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like to investigate in your next CRISPR...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. It can be very time consuming... benefit of working with surface proteins is also that such proteins are easily studied in living cells...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest...screen, each gene is not targeted by only one gRNA, instead many different gRNAs are used for each gene. At...molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different contexts.... Thus, a screen can be used to target surface proteins to determine which contact points are involved...

... E. coli.” ACS Synthetic Biology 4(9) (2015):987-1000. PubMed PMID: 25822415. Find plasmids from this...allowing a spacer-repeat “brick” to be inserted. The BsaI site remains intact, allowing subsequent “bricks...make sure that each cell that gets the plasmid contains all of the desired gRNAs. This increases the chance...the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two U6 promoters. To use ...available from the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods....)-containing plasmids. Each destination vector contains GFP, enabling you to select cells with high GFP...enables users to express up to 7 gRNAs. The kit contains different destination vectors depending on the...

...fibroblasts to myoblasts. Cell, 1987. 51(6): p. 987-1000. PubMed PMID: 3690668. 40. Ferber, S., et al., Pancreatic...transdifferentiation of glucagon-producing α-cells into insulin-producing β-like-cells [37, 38]. In 1987, Davis... ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(4): p. 429-...duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced...

...exctinction coefficient and quantum yield divided by 1000. pKa is the pH at which fluorescence intensity is...Includes many N- and C-terminal fluorescent proteins Insect Sutherland Lab Plasmids - For multicistronic...different fluorescent tags for you to create fusion proteins with your gene of interest. Special... Monomer not a Monomer? Background Fluorescent proteins are genetically encoded tools that are used extensively...have engineered numerous GFP-variants and non-GFP proteins that result in a diverse set of colors. Addgene...different fluorescent tags for you to create fusion proteins with your gene of interest. These are organized... at high concentration, so this is noted here. Proteins that are derived from GFP and contain the A206K...

...interest is expressed. Summary Promoters are about 100-1000 base pairs long and are adjacent and typically upstream...general transcription factor proteins and histones can bind. Histones are proteins found in eukaryotic cells...distal promoter also contains transcription factor binding sites, but mostly contains regulatory elements...transcription factors. Transcription factors are proteins which recruit RNA polymerase and control and regulate...sequences including enhancers, boundary elements, insulators, and silencers. Promoter Regions There are three...is located most proximal to the start codon and contains the RNA polymerase binding site, TATA box, and...is the TSS which is where transcription actually begins. Only eukaryotes and archaea, however, contain ...

...sonication to break DNA up into fragments of 300-1000 bps in length. Note: This sonication process may...four protein chains: two large heavy chains and two small light chains. These four domains come together...their monovalent nature. Darpins - Darpins are genetically engineered small proteins derived from ankyrin ...ankyrin repeat proteins. These are antibody mimics instead of modified antibodies and are not closely related...synthetic binding proteins which use the Fibronectin type III domain as their scaffolding instead of the antibody...Denature proteins, using heat and/or chemicals, and run on an SDS-PAGE gel to separate proteins by size...your antibody’s specificity. A blank (contains water or buffer instead of a biological sample), which captures...

...produces point mutations at a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate.... The Moffat laboratory maintains a website for the TKO library that contains up-to-date information and...(3) but its portability to other bacterial strains remains seriously limited, as prior optimizations are...ligase to biotinylate proximate proteins in the cell. These candidate proteins can be enriched via biotin ...of fusion proteins, requires less biotin, and shows enhanced labeling of proximate proteins. Overall BioID2...optimization of industrial yeast strains. However, industrial yeast strains are more difficult to genetically...which “bait” proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with...

...Maaike Pols, PhD, a Developmental Editor at F1000Research.  With many changes taking place in the world...journal. An increasing number of funding bodies insist that the research data resulting from their funding...your Addgene plasmid #), and other reagents. F1000Research, among many other journals, has joined a pilot...many journals use to verify whether a manuscript contains all the necessary information to be of maximal...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...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile tubes. Store the tubes at -20 °C...PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid... optimized for each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid ...

... organization issues – F1000 just announced the launch of their F1000Workspace.  Give your favorite searching...though you may need to be on a network with an institutional subscription), archived on PubMed Central, or...Check out the hashtag #365papers on Twitter for inspiration and accountability. There are a few different...reprints and papers with scribbled notations in the margins. If you prefer, printing and hand-writing notes...guest blogger! Johnna Roose is a biochemistry instructor in the Department of Biological Sciences at Louisiana...Maaike Pols, PhD, a Developmental Editor at F1000Research  ...

...sector throughout their careers (McDowell et al, F1000Research, 2014; Dolan, F100Research, 2016). These events...students and postdocs broadly – and thus PI’s and institutions in turn. The first is the passing and upcoming...the NIH recommended pay scale – used by many institutions to set postdoc wages. The second event of note...simply being able to categorize all postdocs at institutions, will be challenging. At the same time, there...level, FoR is currently compiling a database of institutional responses to the FLSA ruling. Similarly, from...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...

...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...solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile tubes. Store the tubes at -80 °C... deionized water and adjust to a final volume of 1000 mL. Stir at RT until fully dissolved. Pro-Tip This...For each T-175 flask: Aspirate culture media and rinse once with 10 mL of PBS. Aspirate PBS and add 2 mL... at ~80% confluence. Aspirate culture media and rinse once with 60 mL PBS. Aspirate PBS and add 35 mL ... then transfer the cells into a sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and ...

...and flow cytometry. F1000Research, 13, 817. https://doi.org/10.12688/f1000research.154034.1 Laflamme, ...approach, which focuses on validating antibodies against markers of the human blood leukocyte populations...cases, we would recommend a knockdown approach instead. In this approach, you’re looking for an antibody...synaptotagmin 1 page. The data produced is available on the F1000 gateway and Zenodo community.  Knockdown approach...permanent knockdown, consider using shRNA vectors instead. With both siRNA and shRNA, it can be difficult...induce/suppress the expression of many related proteins, not just your protein of interest, which can ...interest, which can be difficult to find for many proteins. In practice, it was necessary for us to combine...

...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...should be properly disposed of following your institution’s hazardous waste procedures. Last Update: February...February 7, 2022 Protocol Video Watch this instructional video to learn how to affinity purify recombinant... Adjust pH to 7.0 Autoclave or filter sterilize 1000X protease inhibitor cocktail 25 mg leupeptin 50 mg...for your columns, refer to the manufacturer’s instructions. Attach the Gravitrap column to a clamp on a...

...fluorescent proteins. Additional fluorescent protein tools Photoactivable fluorescent proteins (proteins that... Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed...danger. In this post we’ll focus on how fluorescent proteins can and have been used to monitor cellular movements...specimen. It allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated...that have been tagged with different fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation...Cellular trafficking studies enabled by fluorescent proteins The optimization of GFP and its derivatives by...changed everything. For the first time, fluorescent proteins gave researchers the ability to track immune cells...

...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...cloning cylinders. Workflow Timeline This protocol begins with a stable cell pool. For details, see our protocol...homogenized cell solution to 990 µL complete medium. Instead of transferring 0.125 µL, now transfer 100 times...

...) if NHEJ joins the distal ends together. Similarly, it may be possible to direct insertion of an exogenous...Potential. PLoS Genet 5:e1000683 . https://doi.org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P...of breaks) by NHEJ-dependent repair (“pop-in” insertion) provided a template containing compatible overhangs...Renouf B, Renaud J-B, Sallmyr A, Ruis B, Oh S, Tomkinson AE, Hendrickson EA, Giovannangeli C, Jasin M, ...

...you want to have 100x-1000x coverage of your library: in other words, 100-1000x more reads than number... the DNA prep is larger. And since the library contains many plasmids, care must be taken to ensure that...plasmids in the library are identical except for the insert; there’s no need to sequence other parts of the...

..., and Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not...boundaries defined by anti-CRISPR proteins or running into other proteins naturally bound to the DNA. Cas3...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 O’Brien, R. E., Bravo, J...The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA (dsDNA) in what...Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems INTEGRATE: Bacterial...

...Kit Retroviral Barcoding Library (Kit # 1000000097) The Winslow lab barcoding library consists of 96 retroviral... for packaging, however, the isoforms of these proteins, as well as the viral LTRs differ. Therefore, ...Multiple Lentiviral Expression System Kit (Kit #1000000060) The MuLE (Multiple Lentiviral Expression) system...

...model organism, they insert into the host’s genome. Then, the transposon insertion sites are detected with...transposase proteins (TnsA, TnsB, TnsC), and 4) the donor DNA or mini-transposon that contains the DNA cargo... out of the laboratory. F1000Res 9:135. https://doi.org/10.12688/f1000research.21018.1 Munoz-Lopez M, ...and result in human disease. While transposon insertions can be detrimental, it also drives genomic evolution...that moves around. This DNA sequence encodes the proteins the transposon needs for transposition. Transposition...transposition.  Target site: Different transposons insert at different DNA sequences or target sites. Integration...duplication of this target site sequence at the site of insertion.  Types of transposons Transposable elements ...

...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 Additional resources on ...and understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification...bacteria and nearly 100% in archaea, though Class 1 remains largely underutilized compared to Class 2. Class...effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous grasp... genes, Cas1 and Cas2, which are critical for inserting short fragments of foreign DNA into the CRISPR...CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems called...a CRISPR-associated transposase (CAST) that can insert large fragments of DNA. The V-F variants, which...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...solution through a 0.22 µm membrane. Aliquot 500–1000 µL into sterile tubes. Store the tubes at -80 °C...µg of DNA into 500 µL of Opti-Pro SFM. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid ...

...fluorescent signal. Sanger can sequence approximately 500-1000 bases downstream of the known primer region with...website . Insert The insert is the gene, promoter, or other DNA fragment cloned into the MCS. The insert is ...code and provides the instructions to make every protein within an organism. Proteins are made up of amino...segment of DNA which contains several restriction enzyme sites, enabling easy insertion of DNA by restriction...and the inserted gene. The promoter region is required for the generation of RNA from the insert DNA via... the genetic element. For instance, a promoter of interest could be inserted upstream of the luciferase...posts relating to common E. coli lab strains and E. coli strains specialized for protein expression for...