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We narrowed to 490 results for: ARA-2

Showing: 441 - 460 of 490 results

  1. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ... Watch Joanne give her "Not" Networking 101 Talk 2. Tailor every talk to the occasion Don’t just plan...come up with clear and concise answers. This preparation will help you avoid giving only yes or no answers...

  2. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...PubMed PMID: 3459152. PubMed Central PMCID: PMC323600. 2. Chester, Nicholas, and Daniel R. Marshak. "Dimethyl...small difference in size, the fragments should be separated on a high percentage agarose gel (~3%). Note that...

  3. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ... from damage (Abe & Lim, 2024).    Figure 2: A) When proteins (blue) interact with the air-water...RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking: representative micrograph (C)...allowed the authors to determine structures at comparable or higher resolution than using more complex ...buffer additive MgCl2 and the crosslinking agent glutaraldehyde. Adding RvLEAMshort to the sample before plunge...

  4. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...Poloxamer 188 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent Preparation 1 M NaCl/PBS-MK buffer...Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation and loading of the iodixanol...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps...buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of 100...at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume ...need more time, you can alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal...interface of the 60% and 40% gradient (see Figure 2) with an 18 ga needle. Place the first microcentrifuge...

  5. AAV ddPCR Titration

    Type
    Protocol
    ... 95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold ...should decrease by a factor of 2 across the dilutions. In the example below, 2-fold serial dilutions of a ... considered biosafety level 1 but may require BSL-2 handling depending on the insert. Please ensure that...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400) Dilution... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft software...

  6. Lentivirus ddPCR Titration

    Type
    Protocol
    ...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...diploid cells, thus the reason for multiplying by 2. $$V = 2*{copies\ RRE \over copies\ RPP30}$$ Use the viruses...diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete and transfer... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...

  7. AAV Production in HEK293 Cells

    Type
    Protocol
    ...430825, 175 cm 2 Cellstack 5, Corning 3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated...: 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following to the 2 L sterile bottle: 1836 mL deionized... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding...buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM...Cell-Stack (CS5) (Link opens in a new window) (3,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell...flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day...

  8. Molecular Biology Reference

    Type
    Guide
    ...-hsdRMS-mcrBC) Phi80lacZΔM15 Δ-lacX74 recA1 araΔ139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG tonA...mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG trfA...+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ...mrr-hsdRMS-mcrBC) Phi80lacZM15 Δ-lacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG Common...Invitrogen F- mcrB mrr hsdS20 (rB-, mB-) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20 (StrR ) xyl-5 λ- leu ...unique restriction enzyme recognition site (Figure 2). These elements allow for the propagation of the ...elements are summarized in the table below. Figure 2: General structure of a plasmid. Plasmid Element Description...

  9. Fluorescence Titering Assay

    Type
    Protocol
    ...method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh media Day 4...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension into each well of the 6-well...conical tubes, VWR 21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM...

  10. Western Blot

    Type
    Protocol
    ...iBlot 2 PVDF Mini transfer stack. Set the Top Stack to one side and discard the white separator. Keep ...transfer, block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this... Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...Heat block Mini gel tank chamber Power supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker...running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002 20X TBS Tween...immediately or store at -80 °C until ready to use. Section 2: Determine the total protein concentration and prepare...in deionized water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix...

  11. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    ...biology 14.6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure...the ligation junction were represented. Sixteen separate pools were prepared, each with a different complement...applications such as routine cloning or DNA library preparation. However, when a method relies on accurate ligation... 19329793. PubMed Central PMCID: PMC2719376. 5. Barany, Francis. "Genetic disease detection and DNA amplification...PMID: 1986365. PubMed Central PMCID: PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression...PMCID: PMC208194. 8. Luo, Jianying, and Francis Barany. "Identification of essential residues in Thermus...76.2 (1989): 245-254. PubMed PMID: 2753355. 12. Barany, Francis. "The ligase chain reaction in a PCR world...

  12. Finding Your Perfect Job After University

    Type
    Blog Post
    ...experience in cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in...really passionate about during my degree. I loved parasitology. However, when I enquired about roles in this...end of my three months, I fully understood why parasitology roles in the field require experience. Working...beginning of 2010, I explored options for going into parasitology but another major factor held me back; I do ...

  13. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...Nutr, 129 (2009a): 477–484. PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic...Forster, A. C., Skingle, D. C., & Symons, R. H. “Preparation and uses of photobiotin.” Methods in Enzymology...

  14. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...PCR tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ... reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ...normally sterile dH 2 O. To make a 100uM stock of any primer, add a number of µl of dH 2 O equal to the number...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can ...work adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations...72°C. Run 2 μL on a gel to check size and concentration of PCR product. Master Mix Preparation Multiply...(PCR). Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double...

  15. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ... 10 90 2 x 10 8 10 of 2 x 10 8 dilution 90 2 x 10 7 10 of 2 x 10 7 dilution 90 2 x 10 6 10 of 2 x 10 6...6 dilution 90 2 x 10 5 10 of 2 x 10 5 dilution 90 2 x 10 4 10 of 2 x 10 4 dilution 90 2 x 10 3 Pro-Tip...molecules/μL To obtain a solution at 2 x 10 9 molecules/μL: 1.59 x 10 11 / 2 x 10 9 = 79.8X dilution ...your standard curve plasmid (2 x 10 9 stock made in step #1): Volume of 2 x 10 9 stock or previous dilution...stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution ...time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR run: 1.5 h Data analysis: 30 min Equipment ...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...

  16. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select Analyze... Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC)...choose Use Equation . Select the Show R 2 checkbox. Pro-Tip The R 2 of the trendline should be between 0.95...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample...bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray .... Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to...

  17. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    ...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced....brilliantly and irreverently illustrated in the parable of the Geneticist vs the Biochemist, we geneticists...selection allowed me to optimize a range of editing parameters: oligo repair templates with homology arms of... template design The Meyer lab meticulously characterized several aspects of editing. First, they devised...doi.org/10.17912/DDVH-BG64 Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiácovo MP (2013)...

  18. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ... glacial acetic acid 57 mL of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single...Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated phenol-chloroform...100 μL of cold Solution I. Vortex the solution for 2 min or until all bacteria are fully resuspended. Add...pipetting or carefully pouring. Optional: Add 5 μL of 2 mg/mL RNase A to the supernatant in the new tube and... to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous waste - ...: Ethanol Precipitation To your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of...pellet the bacteria before proceeding with DNA preparation. Pro-Tip If your entire overnight culture cannot...

  19. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ...Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv. ...the surface of extracellular pathogens such as parasites or microbes, or to proteins expressed on the surface...containing a mixture of proteins. The proteins are separated based on molecular weight by SDS-PAGE, and are...

  20. Colony Formation Titering Assay

    Type
    Protocol
    ...Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace media with fresh media containing selection...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...the appropriate antibiotic. Incubate the cells for ~2 weeks. All of the cells in the untransduced (negative...disturb the colonies. Count the colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells...stained with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial...conical tubes, VWR 21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM...
Showing: 441 - 460 of 490 results