...barcodes. Rather than continuing to process 100s to 1000s of samples individually, plexWell™ reduces every...advances in technologies that permit large-scale creation and synthesis (“writing”) of longer pieces of ...limited by costly library prep, as each sample requires an individual library. The plexWell™ workflow ...samples from several plates are pooled together to create a single library containing fragments carrying ...

...pieces of roughly equal size, typically between 200-1000 bp. This is to ensure that your downstream analysis...could mean needing to increase crosslinking to counteract the effects of increased sample agitation; a ...such as qPCR or next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions...crosslinking, which uses formaldehyde or UV light to create strong but reversible bonds. This will keep the... necessary for most ChIP experiences, but the increased binding can mask the epitopes antibodies recognize...provides a truly random digestion, but is harsher and requires more optimization. Enzymatic digestion, while ...shorter sonication time could be an opportunity to decrease crosslinking. (Homogenization for a tissue sample...

...proximity ligation." Nature methods 3.12 (2006): 995-1000. PubMed PMID: 17072308.  4. Dieck, Tom, et al. "...neurodegeneration. The level of this protein is decreased in Alzheimer’s disease patient’s brains. However... of radioactive materials is highly regulated, requires designated work areas and come with health risks...

...particles. Studies have found that values can be 10-1000 fold higher than for functional assays depending.... Developing the proper control line typically requires transducing with a limiting multiplicity of infection... How Lentivirus is Used in CRISPR Genome-Wide Screening Learn How Lentivirus Has Been Used for Lineage...

... (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of their target...interest is toxic. To combat this, scientists have created synthetic promoters, which typically include some...binding sites, commonly used in Drosophila Specific Requires the presence of Gal4 gene to activate promoter...Promoter from T7 bacteriophage Constitutive, but requires T7 RNA polymerase. When used for in vitro transcription... Negligible basal expression when not induced. Requires T7 RNA polymerase, which is also controlled by...Promoter from Sp6 bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high ...with somewhat weak expression. lacIq mutation increases expression of the repressor 10x, thus tightening...

...coefficient and quantum yield of the protein, divided by 1000. In many cases the brightness is compared to that...many FPs (particularly those derived from GFP) requires oxygen. Therefore, these FPs cannot be used in... the time it takes the FP to correctly fold and create the chromophore. This can be from a few minutes...

...includes SpCas9, and shortern orthologs approximately 1000 amino acids in size. From the pool of shorter orthologs...and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating ...drawbacks limit the utility of these systems: St1Cas9 requires a very specific PAM sequence that limits the number...efficiency of AAV-SaCas9 in vivo, the Zhang lab created an all-in-one SaCas9 and sgRNA construct using ...histologically normal and liver injury markers were not increased compared to a control AAV-GFP. Not only did the...

... Flp-dependent Cre and Flp-dependent Cre, Flp, and VCre-dependent Constitutive (non-cre-dependent) Clear...dTomato Cre dependent 1, 2, 5, 9, rg* Fishell 100043 pAAV.synP.DIO.EGFP.WPRE.hGH Syn EGFP Cre dependent...None Other Cre-dependent color switch Brainbow constructs Spaghetti monster tags Activity Cre-dependent...EF1a EYFP Cre dependent 1, 2, 5, 9, rg* Deisseroth 28306 pAAV-FLEX-tdTomato CAG tdTomato Cre dependent...nls-EGFP Cre dependent 1, 2, 5, 8, 9, rg* Harvey 131002 pAAV-Ef1a-DIO mScarlet EF1a mScarlet Cre dependent... hSyn EGFP Cre dependent 1, 2, 5, 8, rg* Roth 50459 pAAV-hSyn-DIO-mCherry hSyn mCherry Cre dependent 1...pCAG-FLEX-EGFP-WPRE CAG EGFP Cre dependent 1, 2, 5, 8, 9, rg* Zeng 59331 pAAV-CAG-FLEX-EGFP CAG EGFP Cre dependent 1 Wickersham...

...Blue-wavelength fluorescent protein Activity Cre dependent Flp dependent Cre and Flp dependent Constitutive Serotype...CAG Arch GFP Cre dependent 1, 9 Boyden 28305 pAAV-FLEX-ArchT-tdTomato CAG ArchT tdTomato Cre dependent 5...eNpHR EYFP Cre dependent 9 Deisseroth 26966 AAV-Ef1a-DIO eNpHR 3.0-EYFP EF1a eNpHR 3.0 EYFP Cre dependent...soma-targeted) FusionRed Cre dependent 5 Yizhar 109048 pAAV-CAG-DIO-NLS-mRuby3-IRES-eGtACR1-ST CAG eGtACR1...soma-targeted) mRuby3 Cre dependent 9 Adesnik 137155 pAAV-nEF-Con/Fon-iC++-EYFP nEF iC++ EYFP Cre and Flp dependent...eOPN3 mScarlet Cre dependent 1, 5 Yizhar 139504 pAAV-CAG-DIO-PPO-Venus CAG PPO Venus Cre dependent 5 Bruchas...PHPeB Dimidschstein 100061 pAAV-Ef1a-DIO C1V1 (t/t)-TS-mCherry EF1a C1V1 (t/t) mCherry Cre dependent 9 Deisseroth...

...61727 mCherry-Parkin-IRES-zeo PRKN mCherry Parkinson's Stephen Tait 61728 YFP-Parkin-IRES-zeo PRKN YFP Parkinson's... pAAV-AICD-NLS-IRES-hrGFP APP CMV Alzheimer's Hélène Marie 107544 pAAV-AICD-NES-IRES-hrGFP APP V5 CMV...pLV-EF1a-KIF5A-HA-IRES-Puro KIF5A HA eF1a ALS Rosalind Segal 166953 pLV-EF1a-KIF5AR280H-HA-IRES-Puro KIF5A HA...171786 MSCV-Fbxo7-IRES-GFP FBXO7 GFP Parkinson's Heike Laman 171787 MSCV-Fbxo7(ΔF-box)-IRES-GFP FBXO7 GFP...Lenti-sgAtm#1/Cre ATM CMV Ataxia telangiectasia Charles Swanton 173580 Lenti-sgAtm#2/Cre ATM CMV Ataxia... pAAV-hSyn-GFP-IRES-mSNCA SNCA GFP hSyn Parkinson's Jun Ding 185715 pAAV-hSyn-GFP-IRES-hSNCA SNCA GFP ...pAAV-hSyn-GFP-IRES-mSNCA(1-95) SNCA GFP hSyn Parkinson's Jun Ding 185717 pAAV-hSyn-GFP-IRES-hSNCA (A11P/...

...Low 572/ 610 FP595 Tetramer Green Dark/ Red 70 - 1000 Medium 590/ 600   Choosing a photoactivatable...photoconverts predominantly to the anionic form, causing increased fluorescence when excited with 488nm blue light...which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence when illuminated with blue light...performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry (E26V / ...one since the formation of mEosFP’s chromophore requires a temperature below 30°C and this is not ideal...photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't become...the absence of photoactivation resulting in an increased signal to noise ratio. PA-FPs with higher contrast...

... E. coli.” ACS Synthetic Biology 4(9) (2015):987-1000. PubMed PMID: 25822415. Find plasmids from this...plasmid contains all of the desired gRNAs. This increases the chance that all the edits you want to make... cleave outside of their recognition sequence, creating flanking overhangs. These overhangs can be customized...Expression Systems (MuLE) Kit: This kit can be used to create lentiviral vectors expressing wt humanized Cas9...the gRNAs. gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs...using 3 distinct spacer-repeats, you could easily create 7 unique arrays (e.g. for spacers A, B, and C, ...is possible with efficiency >70%. CRMAGE is an incredibly fast protocol, with only 5 hours incubation time...

... Mammalian shRNA Tools for RNAi! Cre-Lox technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral ...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can be...the pLKO.1 vector can be found here. Cre-Lox technology Puro.Cre empty vector (Plasmid #17408)   This ...of mouse transgenics. Experimental uses include: Cre-dependent gene expression, gene knockout, and selection... vector deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. ...shRNA), mutation/editing (CRISPR-Cas9) or deletion (Cre), together with expression of fluorescent or enzymatic...CRISPR pooled libraries that allow functional screening of thousands of genes in genome-wide experiments...

...sonication to break DNA up into fragments of 300-1000 bps in length. Note: This sonication process may...cytometry requires a negative control, a small subset of the sample with no antibodies. It also requires single-antibody... cell cultures using hybridomas, cell cultures created by fusing B cells with myeloma cells (immortalized...immortalized B cells). The genetic reorganization that creates variety in the antibody allowing for their extreme...animals immunized as described above are used to create hybridomas, which produce large amounts of a single...recombinant antibodies. Recombinant antibodies are created by cloning antibody genes from specific hybridoma...goes through a randomized reorganization process, creating a unique binding site. This process, known as ...

...pAAV.CMV.HI.eGFP-Cre.WPRE.SV40 Cre Recombinase James M. Wilson AV-9-PV2394 107788-AAV9 AAV.rTH.PI.Cre.SV40 Cre Recombinase... Cre Recombinase James M. Wilson AV-5-PV2408 105550-AAV5 pAAV.GFAP.Cre.WPRE.hGH Cre Recombinase James ...AAV.TBG.PI.Cre.rBG Cre Recombinase James M. Wilson AV-8-PV2004 105545-AAV8 pAAV.CMV.HI.eGFP-Cre.WPRE.SV40 Cre Recombinase... 0.4.Cre.SV40 Cre Recombinase James M. Wilson AV-1-PV2676 105553-AAV1 pENN.AAV.hSyn.Cre.WPRE.hGH Cre Recombinase...pAAV.CMV.HI.eGFP-Cre.WPRE.SV40 Cre Recombinase James M. Wilson AV-5-PV2396 105558-AAV5 pENN.AAV.CamKII 0.4.Cre.SV40 Cre...James M. Wilson AV-1-PV1090 105537-AAV1 pENN.AAV.CMVs.Pl.Cre.rBG Cre Recombinase James M. Wilson AV-1-PV1848...PV1848 105540-AAV1 pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40 Cre Recombinase James M. Wilson AV-1-PV2004 105545...

...loxP-stop-loxP cassette into the Rosa26 locus, allowing for Cre-dependent expression of a GMAP inserted gene. The...produces point mutations at a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...and later create stable cell lines expressing it. The Church lab fixed this issue by creating a new dCas9...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...CRISPR knockout library and used this library to screen for genes involved in West Nile Virus (WNV) induced...

...Maaike Pols, PhD, a Developmental Editor at F1000Research.  With many changes taking place in the world...world of science publishing, and an ever increasing number of journals and publishers to choose from, it... paper for publication in a scientific journal requires several considerations. First, it is important...them needing a subscription to the journal. An increasing number of funding bodies insist that the research...your Addgene plasmid #), and other reagents. F1000Research, among many other journals, has joined a pilot...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...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...likelihood of multiple integration events decreases as the dilution increases. Calculate the transduction units...cell lines and selection markers. Note this assay requires staining resistant colonies with crystal violet... remove cells and debris. Lentiviral titer can decrease during cycles of freeze-thaw. If you are freezing...

...and flow cytometry. F1000Research, 13, 817. https://doi.org/10.12688/f1000research.154034.1 Laflamme, ...antigen of interest in the given protocol. This requires use of one of the five pillars of antibody validation...validation, and it is recommended to screen multiple candidate antibodies when possible. If cost is prohibitive...synaptotagmin 1 page. The data produced is available on the F1000 gateway and Zenodo community.  Knockdown approach...interest. By correlating your data sets, you can increase confidence that the labeling observed represents...experiment. We find it useful to start with a screening procedure in easily genetically modified cell ...

...primers to create 200-300 bp sequencing products using PCR. A diagram of the product we created for the ...you want to have 100x-1000x coverage of your library: in other words, 100-1000x more reads than number...plasmid. However, lots of library DNA is needed for screening experiments, so the scale of the DNA prep is larger...rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth distribution” curve, or Lorenz...a powerful research tool, but their proper use requires careful amplification and maintenance of good ...wishes you luck in your future library use and screening, and if you have any tips, input, or stories of...

...Systemic capsids packaging Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse...with appropriate cell type-specific promoters or Cre lines. Spatial transcriptomics suggests that AAV-...Huber, N., Pasca, S. P., & Gradinaru, V. (2016). Cre-dependent selection yields AAV variants for widespread...Deverman, B. E., & Gradinaru, V. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting...have developed techniques like CREATE (Deverman et al., 2016), M-CREATE (Ravindra Kumar et al., 2020),...Research in Neurobiology, 4, 100086. https://doi.org/10.1016/j.crneur.2023.100086. Challis, R. C., Ravindra...

...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 O’Brien, R. E., Bravo, J...part of the Class 1 CRISPR family, which means it requires multiple Cas effectors to carry out genomic editing...different compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade...Figure 2: Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex,...Tomoji Mashimo lab and the Tilmann Weber lab — to increase efficiency of these systems, though consistent...General overview of the Cascade-Cas3 mechanism. Created with BioRender.com.   The reeling mechanism...unwinding of dsDNA occur simultaneously, which creates a loop in the target strand that is cut occasionally...

...useful tool for marking and tracking cells is the the Cre/lox recombination system. A researcher can flank ...expression is turned on or off in cells expressing Cre. The result of a system like this is a mosaic of ...respectively. The permanent labelling of cells using cre-lox is advantageous when compared to photoactivable... In the past 15 years efforts have been made to create transgenic mice expressing fluorescent protein ...auto-fluorescence (Drobizhev et al. 2011). But this requires other lasers than the standard Ti:Sapphire laser...wavelength as it has been shown that even a small incremental change of the excitation wavelength can significantly...called Brainbow, the Ubow mouse strain has been created to fate map Langerhans cells and follicular dendritic...

... Zinc Finger Consortium: OPEN Reagents (Kit # 1000000013 ) Depositing Labs: Keith Joung This kit consists...17. PubMed . Description Use of the OPEN method requires OPEN pools (available by request from the Joung...paper . How to Cite this Kit These plasmids were created by your colleagues. Please acknowledge the Principal...Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods...publication was a gift from Keith Joung (Addgene kit # 1000000013)" For your Reference section: Oligomerized pool...

...breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 Additional resources on ...systems has led to extreme sequence divergence that requires a multi-pronged analysis to sort effectively. ...type to subtype to variant (shown in italics). Created with BioRender.com.   Class 1 versus class ...underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class 1 CRISPR systems are less popular...Cascade. As you might imagine, this is handy for creating large genomic deletions. Type I systems are also...with Cas3 (pink). Complex shown is from type I-E. Created with BioRender.com.   Type III Type III is ...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette...enough master mix for all samples). Each sample requires 15 μL of master mix. Pro-Tips Use a "Universal...95 uL 20X 200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution 4 20 uL Dil. 3 80 uL 5X 5000X Dilution ...indicates the presence of primer dimers which can increase background signal and alter the Ct values of your...

... ligase The Roux Lab in 2016 optimized BioID to create BioID2, a smaller and improved promiscuous biotin..., BioID2 exhibits enhanced proximal labelling, requires less biotin, and allows scientists to more selectively...was its low sensitivity. Sensitivity could be increased with higher expression levels but for many fusion...until they identified a mutant with significantly increased sensitivity that did not affect the function or...miniTurbo were developed in the Ting Lab in 2018 to increase the speed of proximity labeling by promiscuous...APEX2, although it can rapidly label proteins, requires H2O2 which can be toxic to living cells. To circumvent... BASU exhibits 1,000 fold faster kinetics and increases the signal to noise ratio 30-fold compared to ...

...used for large scale edits. These proteins, like Cre recombinase or phage derived serine integrases, insert... GAT; increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease...others work to increase Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes...backbone HypaCas9 - increase Cas9 proofreading and discrimination evoCas9 - decrease off-target effects...domains to expand PAM recognition, increase specificity, and decrease off-target effects Sniper-Cas9 - ...compatible with truncated gRNAs to increase specificity SuperFi-Cas9 - increases fidelity with reduced nuclease...integration of donor DNA. Genome-Wide Screens Using CRISPR In a genetic screen, a population of cells with mutations...

...C-terminal fluorescent protein tags that can be used to create fluorescent protein fusions with your protein of...expression of a recombinant protein with a 6x His tag. Requires a strain with T7 RNA polymerase. Amp resistant...in response to green light. BIOFAB plasmid set 1000000037 N/A N/A Drew Endy This kit contains plasmids ...expression from that element. When this activation requires a transcription factor to bind to a small molecule...heterodimer formation with dominant negative AraC decreases GFP expression. Zinc Finger Consortium: Modular...strain for performing a bacterial two-hybrid (B2H) screen of modularly assembled zinc fingers. Return to ...

...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ...entry clone and destination vector creates two products: the desires expression clone and a byproduct containing...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain...Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination of an attB-flanked...attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the insert into an...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the inset into...vector will recombine with the attL-entry clone to create the expression clone.    Figure 6: Generating...