...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...ensure that you are in compliance with your institution’s biosafety regulations. Last Update: February... °C. See below for other Reagent Preparation instructions. Wipe down all pipettes and equipment with 10%...suggest preparing fresh solutions after one month. 1000X protease inhibitor cocktail 25 mg Leupeptin 50 mg...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Pipette controller Pipette...your cell pellet but will generally be between 250–1000 µL . The ideal lysis buffer will vary depending ...weight proteins and higher percentage acrylamide when immunoblotting low molecular weight proteins. This...Transfer for 5–6 min for proteins <30 kDa. Transfer for 8–10 min for proteins >150 kDa. Select Start Run...Introduction Western blot is a technique used to separate proteins by size followed by detection using antibodies...Incubate with secondary antibody Video Watch this instructional video to learn how to use western blotting to... Before Starting Refer to the manufacturer's instructions for additional information specific to your ...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Pipette controller Microcentrifuge...Pipette tips, 200 µL, VWR 76322-150 Pipette tips, 1000 µL, VWR 76322-154 Pipettes, 5 mL, VWR 89130-896 ...1 or later: Image analysis Video Watch this instructional video to learn how to determine purity and concentration...microcentrifuge. Remove the gel from the plastic wrapper and rinse with deionized water. Gently remove the white sticker...and secure it. Note: Check the manufacturer’s instructions if you are unsure of the correct orientation...the gel. Carefully remove the comb from the gel. Rinse each well with 200 µL 1X MOPS running buffer. Fill... a plastic tray with 100 mL of deionized water. Rinse gel with deionized water for 5 min with gentle agitation...

...Research in Neurobiology, 4, 100086. https://doi.org/10.1016/j.crneur.2023.100086. Challis, R. C., Ravindra...exists to prevent substances in the blood, like proteins and viruses, from crossing into the brain. While...increase the chances of inducing an immune response against the capsids or transgenes. Sensory neurons and ...as the type of transgene delivered: fluorescent proteins, optogenetic sensors, CRISPR elements, etc... ...capsids express differently in different mouse strains, a phenomenon that we are calling “strain-specific... but is more effective than AAV9 in many other strains, including FVB/NCrl, 129S1/SvImJ, DBA/2J, and C57BL...table from Huang et al, 2019 for affected mouse strains). However, other capsids cross the BBB via interactions...

...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...protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected ...indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) against a purified antigen to validate an antibody. This... the antigen and how to probe with an antibody against the target to show a dose response. Sharing speeds...several days by incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes...minutes. Absorbance was read at 450 nm and plotted against the micrograms of antigen loaded. Last reviewed...

...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...

... Alzheimer's disease & Parkinsonism. https://doi.org/10.4172/2161-0460.1000439 6. Wu et al. 2020. . “Advances...in monoallelic diseases In 2013, a team led by Jinsong Li from the Chinese Academy of Sciences found that...independent team led by Hans Clevers at Hubrecht Institute in The Netherlands used the CRISPR/Cas9 genome...handle on how cleavage sites are repaired.” Those insights will be critical for scientists’ ability to influence...addition, the current work in developing anti-CRISPR proteins aid the limitation of these mutations, to control...

...were at institutions focused only on tracking hours, and 22% of postdocs were at institutions where no... of some public institutions) to cancel plans to raise salaries. What are institutions doing after the...planning unionization at their institutions. One clear message that institutions have sent, with the lack of...their institution is doing from Future of Research over social media, not from their institution), is ...raise were: postdocs at institutions like Stanford and the Whitehead Institute, where salaries had already...street from these institutions) are raising salaries. Postdocs at the Salk Institute in La Jolla California...This post discusses how a nationwide injunction against the FLSA is affecting universities' decisions to...

...Blast 1000000173 Knockout Mouse Liu 3rd 10 185,836 Mouse GeCKO v2 1000000052 (1 plasmid) 1000000053 (2 plasmid...Varies Bassik Mouse CRISPR Knockout Library 1000000121 — 1000000130 Knockout Mouse Bassik 3rd 10 Varies Berger...83978 1000000091 Activation Human Weissman 3rd 5 10 104,540 209,080 CRISPRa-v2 83996 1000000093 Activation...83969 1000000090 Inhibition Human Weissman 3rd 5 10 104,535 209,070 CRISPRi-v2 83987 1000000092 Inhibition...Birsoy 3rd 8 20,051 Human GeCKO v2 1000000048 (1 plasmid) 1000000049 (2 plasmid) Knockout Human Zhang 3rd...10 12,472 SAM v1 - 3 plasmid system 1000000057 (Zeocin) 1000000074 (Puromycin) Activation Human Zhang ...178,896 Activity-optimized genome-wide library 1000000100 Knockout Human Sabatini and Lander 3rd 10 187,535...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette...calculate the titer for each sample: $$T = {R*C*D*1000\over V}$$ Where: T = Titer, GC/mL R = Reaction volume...depending on the insert. Please ensure that you are in compliance with your institution’s biosafety regulations... To ensure that the droplets are not disrupted, insert the pipette tips directly in the center of the ...Pro-Tip To ensure that the droplets are not disrupted insert the pipette tips and gently touch the bottom of...

... to train our young scientists for futures both inside and outside of academic research (4). There are...industry, regulatory, or clinical environment. It remains contested, however, whether a better solution would...University, has recently published an article arguing against reducing the number of graduate students accepted...grassroots organisations that represent postdocs and institutes that employ them, the diversity of trainee backgrounds...Research: a perspective from junior scientists." F1000Research 3 (2014). Pubmed PMID: 25653845. Pubmed Central...

...limited to traditional restriction enzyme cloning. Instead, you can choose a molecular cloning technique that...takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction... inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined...vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO cloning adds...Method B to create an entry clone: TOPO cloning the insert into an attL-containing entry vector.   Method...interest and a attL-entry vector. This fragment is inserted in a multiple cloning site (MCS) of an attL-containing... create an entry clone: Restriction cloning the inset into an attL-containing entry vector.   Pro...

...within living cells. By coupling fluorophores to proteins, pioneers of this technique were able to directly...FRET-based biosensors, and interactions between DNA and proteins. FRET, in theory, is an easy technique, but it...to studying an unknown interaction between two proteins, these parameters are difficult to overcome but...FRET_teaching_module.pdf Day RN, Davidson MW. Fluorescent proteins for FRET microscopy: monitoring protein interactions...Bioessays. 2012 May; 34(5):341-50. doi: 10.1002/bies.201100098. Sekar RB, Periasamy A. Fluorescence resonance...

...interactions as well as be used for both soluble and insoluble proteins, which can be a challenge for classic antibody...can take hours to label proteins and APEX2, although it can rapidly label proteins, requires H2O2 which ...interacting proteins in one simple experiment. PCA is an in vivo technique where two proteins are fused ...interaction of two proteins while also biotin labeling and purifying additional proteins from a complex of... and an RNA component that contains an RNA motif of interest. Any proteins that bind the RNA motif will...interacting cell surface proteins with a biotin tagged LPETG peptide. Biotin tagged proteins can then be purified...There are approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells...

...al., 2002 used an overlapping vector with 440 or 1000bp overlaps to deliver the alkaline phosphatase gene...transgene.The AAV encoding part A of the transgene contains a promoter and the 5’-fragment of the transgene...donor. The AAV encoding part B of the transgene contains a splice site acceptor followed by the 3’-end ...methods. The AAV encoding part A of the transgene contains the promoter, the 5’ half of the transgene, a ...homology. The AAV encoding part B of the transgene contains the region of homology, a splice acceptor sequence...help prevent the accumulation of such truncated proteins. Examples of use:  Ghosh et al., 2008 used a...directed repairs (HDR) with CRISPR/Cas9. Donor A contains: 400bp homology arms to the site in the genome...

...occurring E. coli plasmid ColE1 (Goebel & Helinski, 1968; Bazaral & Helinski, 1968), in which the plasmid was ...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore... of plasmids, taking many forms (Levene, 2009; Higgins & Vologodskii, 2016). Plasmid multimerization occurs...to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats (Ribeiro...et al., 2009). Since multimers have additional origins of replication compared to monomers, they are also... of multimer present in your sample. In these instances, please note that the supercoiled dimer band can.... If the presence of a multimer in your sample remains unclear after visualizing it on a gel, one of the...

... well characterised antibodies against all human extracellular proteins with community-based validation...18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end...have a reproducibility “crisis” on our hands and instead provide a brief conceptual framework for the causes...ll see in Steve Almo’s talk about work at the Institute for Protein Innovation (IPI) (49:45 - 1:18:45)...while FPbase curates information about fluorescent proteins so you can best apply these technologies to your.... Finally there are new publishing outlets like F1000, PeerJ, PLoS, and bioRxiv that make it easier to...formal systems, individual labs can certainly institute better experimental design practices including...

...optogenetics AAV collection, including channelrhodopsins, halorhodopsins, and more....GFP Syn ChR2/H134R GFP Constitutive 8, rg* Boyden 100054 pAAV.CAG.hChR2(H134R)-mCherry.WPRE.SV40 CAG ChR2...] CAG ChrimsonR tdTomato Flp dependent 8 Boyden 100049 pAAV.hSynap.ChETA(E123T/H134R)-eYFP.WPRE.hGH Syn...Syn ChETA EYFP Constitutive 1, 9 Deisseroth 100050 pAAV.CaMKIIa.ChETA(E123T/H134R)-eYFP.WPRE.hGH CaMKII... EYFP Constitutive 1, 5, 9, PHPeB Dimidschstein 100061 pAAV-Ef1a-DIO C1V1 (t/t)-TS-mCherry EF1a C1V1 (...

...when using highly competent cells. If you used 100-1000 ng of total DNA in a ligation you will often get...competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock...many genetic modifications to create bacterial strains that can be more easily transformed and that will...efficiency, we recommend that you follow the instructions that came with your competent cells. Pro-Tips...out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate...be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. ... the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing ...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...ensure that you are in compliance with your institution’s biosafety regulations. Workflow Timeline Day...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Reagents 1X PBS Microcentrifuge...staining cells in culture with a primary antibody against a target protein and a fluorescent secondary antibody...labeling cells in culture with a primary antibody against a target protein and a fluorescent secondary antibody...need to be optimized for different cells, target proteins, etc. Sharing speeds science. We believe that ... Before Starting Refer to the manufacturer's instructions for additional information specific to your ...Paraformaldehyde fixation works well for many target proteins, it may not be the best fixation method for all...Remove the paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of...

... (Kit # 1000000013 ) Depositing Labs: Keith Joung This kit consists of plasmids and strains used to ... packaged plasmids and 3 individually packaged strains, which are shipped as bacterial stabs at room temperature...publication was a gift from Keith Joung (Addgene kit # 1000000013)" For your Reference section: Oligomerized pool...

... control AAV collection, including fluorescent proteins, spaghetti monster, and more....Browse our control AAV that express fluorescent proteins under general promoters. These AAV can be used... Constitutive 2, 5, 8, 9, rg*, PHP.eB Gradinaru 131000 pAAV-CaMKIIa-mScarlet CamKIIa mScarlet Constitutive...Dlx dTomato Cre dependent 1, 2, 5, 9, rg* Fishell 100043 pAAV.synP.DIO.EGFP.WPRE.hGH Syn EGFP Cre dependent...

...middle is hundreds, and the third is tens. Therefore, 1000 µL would read as 100 from top to bottom (as shown...Anatomy of a Pipette This front view photo of a P1000 pipette shows the plunger at the top, which is used...tip off of the pipette. This side view photo of a P1000 pipette shows the volume adjustment ring right below...come in standard sizes: P2, P10, P20, P200, and P1000. The table shows the pipette with the appropriate...This image shows the P2, P10, P20, P100, P200, and P1000 as shown from left to right. Pipette Dispense Volume... 2 to 20 µL P100 10 to 100 µL P200 20 to 200 µL P1000 100 to 1,000 µL Each pipette should be used with... dispense the correct amount of liquid reagent. P1000: The first red digit is thousands of µL, the middle...

...comprehensive human transcription factor collection that contains 1,836 genes encoded by 3,548 isoforms and two ...scRNA-seq (PDF, 59.2 KB) Plasmids ID Plasmid Gene/Insert Pooled Library The MORF pooled library is a comprehensive...ID PI Description MORF library 137000, 192821, 1000000218 Zhang A collection of human transcription factor...

...commonly used adenovirus terms Adenoviral Strains Bacterial strains for generating adenovirus. ID Strain Description...beta-gal Shuttle Test plasmid that contains β-gal and GFP; contains ∼10Kb genomic DNA stuffer Vogelstein...Kit Description PI 1000000176 AdenoBuilder toolkit Plasmids contain modular inserts for seamless assembly...Collection of adenovirus shuttle vectors and strains Viral...Vogelstein 16399 AdEasier-1 BJ5183 derivative that contains AdEasy-1™ plasmid Vogelstein Adenoviral Plasmids...database for adenoviral transfer plasmids that have inserts. ID Plasmid Type Description PI 16400 pAdEasy-1...recombining shuttle plasmids; for use with large inserts Vogelstein 179201 pAd5-B6 ∆E3-GFP Adenoviral A ...

...pipettes Ice bucket to hold antibiotic Antibiotic at 1000 x concentration disolved in the appropriate liquid...use. Negative Result 1: Both Strains Grow Assuming the appropriate strains were streaked on the appropriate...your strains are no longer viable. You can check for this possibility by streaking out both strains on ...antibiotic to the molten gel mix, you should create a 1000x stock solution. For example: If you’ll be preparing...slightly. Even so, you should always use thermally insulated gloves when removing anything from the autoclave...appropriate plates, then if both strains grow, it’s possible that: The antibiotic broke down. You forgot to...concentration for selection. Negative Result 2: Neither Strains Grows If neither strain grows, it's possible that...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette...titers were similar to those reported by these institutions. This protocol is for a 96-well plate with 20...run: 1.5 h Data analysis: 30 min Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel...Pro-Tips Use a "Universal" SYBR master mix which contains a high-quality DNA polymerase and a blend of dTTP...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 ...the final volume (mix with >50 uL if your well contains 100 uL) Use a multichannel pipette to load the...tube. Run the following protocol in your qPCR instrument using SYBR detection: 98 °C 3 min / 98 °C 15 ...

... System Contains 336 pFusX clones allowing for Golden Gate assembly of 15-16 RVD TALEN inserts in one ...plasmids of pre-assembled trimer and tetramer RVD domains designed for rapid TALEN assembly. Kiran Musunuru...message. Both vectors utilize homodimeric FokI domains. 38143 pC-GoldyTALEN Golden Gate TALEN 2.0 36033...sequences within core promoter regions. pTAL5-BB contains the GAL1 promoter, placing TALORs built into this...under galactose-inducible expression. pTAL6-BB contains the TEF1 promoter, giving constitutive expression...mutation induction when using TALENs. The FokI domains (DD, RR) used are obligate heterodimers that require...require cloning of left and right TALEN monomer proteins into opposite vectors. 37276 pCS2TAL3-RR 48636...

...inactive “dead” Cas9 (dCas9) contains mutations in both nuclease domains, D10A and H840A, resulting in...naturally-occurring Acr (Anti-CRISPR) proteins can help control CRISPR activity. Acr proteins have evolved in phages ...bidirectional insertions — meaning they may not be suitable for applications where orientation of the insert is ...exhibit almost unidirectional insertions but are more prone to off-target insertions. And in the case of both... edits. These proteins, like Cre recombinase or phage derived serine integrases, insert donor DNA at “...used to install the LSR Bxb1’s landing site into the genome at a desired locus, then Bxb1 installs the donor...and antibody-activator effector proteins; long-term imaging of proteins in living cells (Figure 9B) dCas9...

...Plasmids 101: E. coli strains for protein expression Plasmids 101: Common Lab E. coli Strains Why Bacteria? E...Photoswitchable fluorescent proteins Various PIs Fluorescent protein fusion: contains empty vectors with N and...Expression: contains mEos2 with an N-terminal His tag. Photoactivable fluorescent proteins like this one... 3OC6HSL (Quorum sensing molecule) Cynthia Collins Contains luciferase inducible by the quorum sensing...green light. BIOFAB plasmid set 1000000037 N/A N/A Drew Endy This kit contains plasmids with various combinations...factor bind to a particular sequence? Do these two proteins interact? What happens when I overexpress this...From recombineering to CRISPR , this collection contains a variety of tools to help you harness bacterial...

...Browse our AAV that express fluorescent biosensor proteins, including calcium sensors, under the control ...Cre dependent 5, 9 Tian Calcium Sensor: Twitch2B 100040 pAAV.hSyn1.Twitch2B.WPRE.SV40 Syn Twitch2B none...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette...modified for other targets. Primers and a probe against the cellular ribonuclease P/MRP 30 kDa subunit ...ensure that you are in compliance with your institution’s biosafety regulations. Last Update: July 7, ...well untransduced (add 150 µL of DMEM complete instead of a viral dilution). Remember, this is an additional... to the GeneJet Genomic DNA Purification Kit instructions. Determine the concentration of each sample ... To ensure that the droplets are not disrupted, insert the pipette tips directly in the center of the ... To ensure that the droplets are not disrupted, insert the pipette tips and gently touch the bottom of...

...GFP CMV Parkinson's L. Miguel Martins 14122 pcDNA3-HtrA2-EGFP S306A HTRA2 GFP CMV Parkinson's L. Miguel...Miguel Martins 14124 pcDNA3-delta133HtrA-EGFP HTRA2 GFP CMV Parkinson's L. Miguel Martins 14126 pET20b-delta133HtrA2...Miguel Martins 15939 pcDNA3-HtrA2-FLAG S142D HTRA2 Flag CMV Parkinson's L. Miguel Martins 15940 pcDNA3-HtrA2...Flag CMV Parkinson's L. Miguel Martins 16153 pET20b-delta133HtrA2 S142A HTRA2 His T7 Parkinson's L. Miguel...Miguel Martins 16154 pET20b-delta133HtrA2 S142D HTRA2 His T7 Parkinson's L. Miguel Martins 16155 pET20b-...His T7 Parkinson's L. Miguel Martins 16156 pET20b-delta133HtrA2 S400D HTRA2 His T7 Parkinson's L. Miguel...Miguel Martins 16157 pET20b-delta133HtrA2 A141S HTRA2 His T7 Parkinson's L. Miguel Martins 16158 pET20b-...

...inactive “dead” Cas9 (dCas9) contains mutations in both nuclease domains, D10A and H840A, resulting in...naturally-occurring Acr (Anti-CRISPR) proteins can help control CRISPR activity. Acr proteins have evolved in phages ...bidirectional insertions — meaning they may not be suitable for applications where orientation of the insert is ...exhibit almost unidirectional insertions but are more prone to off-target insertions. And in the case of both... edits. These proteins, like Cre recombinase or phage derived serine integrases, insert donor DNA at “...used to install the LSR Bxb1’s landing site into the genome at a desired locus, then Bxb1 installs the donor...and antibody-activator effector proteins; long-term imaging of proteins in living cells (Figure 9B) dCas9...

... or more from the same tube, but unlike using a p1000 tip it will give you more control of where you’re...LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl solution...downstream applications. To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can try ...

...purification and isolation of cells, organelles, lipoproteins, and macromolecules. Importantly, iodixanol ...bring up the volume with formulation buffer. Use a P1000 to the bottom of the filter and pipette up/down ...of contaminants in each fraction. * AAV capsid proteins VP1, VP2, VP3; M protein marker. Image adapted...