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Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

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We narrowed to 492 results for: ara-2

Showing: 461 - 480 of 492 results

  1. 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...

  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...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...

  3. 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...

  4. 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...

  5. 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...

  6. 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 ...

  7. 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...

  8. 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...

  9. 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 ...

  10. 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...

  11. 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)...

  12. 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...

  13. 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...

  14. 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...

  15. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...Reagent Preparation 1 M sodium phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH 7.0 138 g NaH 2 PO ...dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave...monobasic monohydrate (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to...recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the concentration ...antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type...conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ... channel pipette 20–200 µL single channel pipette 2–20 µL single channel pipette Reagents Aspirating pipette...

  16. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs Watch the...Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water...cloning, and titering and testing your viral preparations. Addgene...Learn about selecting and using a centrifuge to separate different components in a liquid sample Using ...restriction enzymes Agarose Gel Electrophoresis Separate DNA by size on an agarose gel Watch the Video!...Protocols for titering and testing your virus preparations. Name Description Link to Video General Transfection...Ultracentrifugation Purify adeno-associated virus from a preparation produced in mammalian cells Watch the Video! ...

  17. 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 ...

  18. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...concentrations of 0.7% to 2% depending on the size of bands needed to be separated - see FAQs below . Simply...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds...allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases... typically used to separate 5 - 500 bp fragments. How do you get better separation of bands? If you have...electrophoresis, including tips to improve resolution and separation of bands. Protocols...electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for ...

  19. Kit Free RNA Extraction

    Type
    Protocol
    ...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl...sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at 12,000 × g at 4°C to separate RNA from the....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...aliquots of it and storing those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells...sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate. Pro-Tip...

  20. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...materials D.2 Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol...Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty...VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing Oligos...oligo 5 μL Reverse oligo 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick...buffer for EcoRI 1 μL EcoRI 14 μL ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting... For a standard T4 ligation, mix: 2 μL annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning ...
Showing: 461 - 480 of 492 results