Skip to main content

We narrowed to 634 results for: des.2

Showing: 581 - 600 of 634 results
  1. Five Popular Model Organisms

    Type
    Blog Post
    ...variety of environmental conditions, and double every 2 hours. Yeast are also the first eukaryotic genome ...difficult and time consuming. GAL4/UAS was first described in 1993 by Norbert Perrimon’s lab and has been...
  2. 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...0.05% Trypsin/EDTA. Wait ~2-3 minutes for cells to detach. Gently tap the sides of the CS2 to help detach...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...
  3. 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 ...
  4. Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs

    Type
    Protocol
    ...protocol provides information for both biosafety level 1 (BSL-1) and biosafety level 2 (BSL-2). The BSL...potential accidents such as spills. BSL-2 is different because it includes labs that work with agents associated...associated with diseases in healthy humans. BSL-2 includes all of the precautions needed in BSL-1, however...BSL-1 and BSL-2 labs. Protocols... Protocols Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs Personal ...Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs Intro to the Lab Bench Check out more protocols ...and/or face shields can be used as needed. For BSL-2 work always wear glasses/goggles in addition to the...
  5. General Transfection

    Type
    Protocol
    ... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high... for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace...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... use. Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000...times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 ...
  6. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ... from damage (Abe & Lim, 2024).    Figure 2: A) When proteins (blue) interact with the air-water... A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm single ...cryo-EM samples from damage at the AWI. Plus, tardigrades!  Find RvLEAMshort here!    Abe, K. M., & Lim...
  7. 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...
  8. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...ask new and interesting questions in your own lab. 2. Seek multiple mentors during your postdoc At the ...for and how can you do it well? Below, I briefly describe some tips for a successful postdoc fellowship....typically writes the grants that fund your research, guides the research directions of the lab, gives you constructive...Thesis committee members provide expertise in designing and interpreting experiments, as well as career... there a tractable question or are you simply describing a phenomenon? Exploration can be important, but...presentations benefit from practice. If you simply recycle slides from the last time you gave a presentation to a...story I can tell with my data?  Can I make better slides that more effectively tell a story? Generally,...
  9. Lentivirus Production

    Type
    Protocol
    ... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided...packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace...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...culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three transfection...plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives...
  10. 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...
  11. 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...every item I tested was constantly tracked with barcodes and spreadsheets. It was great to be in the lab...industry positions. There were, however, some downsides to working in a large commercial company. In my...aspects of the company and gave me the freedom to design new projects and grow in the role. I understand... job different every day. I love the freedom to design and develop my own projects, but working remotely...
  12. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ...Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv. ...been infected with a microbe, to trigger immune cascades that clear these infections. Anything that generates...immune response and initiates different immune cascades. In the research setting, antibodies of different...antibody, which recognizes the primary antibody and provides the method of detection. You’ll also want to use...used for. Addgene’s Antibody Plasmid Collection includes plasmids that express antibodies, nanobodies, ...
  13. Western Blot

    Type
    Protocol
    ...surface of the iBlot 2 Gel Transfer Device. Close the lid of the device. Select the desired method and make...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...
  14. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps...7.4 1X PBS-MK buffer 100X Poloxamer 188 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent...PBS-MK 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...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 ...(C) (formulation buffer) Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation ...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...
  15. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ... Anti-Desmin [D7]. Plates were incubated with HRP-linked anti-Human IgG secondary antibody for 2 hours...vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: Primary antibody incubation Day 4...with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...serial dilutions of the purified antigen as follows: 2 ng/µL : Add 100 µL of 20 ng/µL stock into 900 µL PBS...microfuge tube and vortex. 1 ng/µL : Add 450 µL of 2 ng/µL stock into 450 uL PBS in a microfuge tube and...37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween...incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section...
  16. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...Bachelor's degree Generally a short-term position (1-2 years) Often a gap-year position taken to transition... are more involved in the science, experimental design, and execution. Can lead to an independent project...advice Give yourself permission to pursue your own desired path. Careers in science are also not limited to...
  17. 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...
  18. 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...for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...
  19. 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...end-joining, but will also efficiently ligate many undesirable structures, including substrates containing gaps...fidelity") better than T4 DNA Ligase (21, 25, 26). Despite this higher fidelity, Taq DNA Ligase can still ...possible NN pairs in the template. The probes were designed such that each possible pairing resulted in a ... high fidelity ligase, along with careful probe design and reaction condition optimization, will be critical...
  20. 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...Last Update: Feb. 8, 2018 Equipment Desktop microcentrifuge Desktop vortexer Vacuum (optional) Reagents...
Showing: 581 - 600 of 634 results