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ExoDNAUC™ Circulating and Exosome-associated DNA Extraction Kit (Urine/Cell Media, 20 reactions)

Details for Product No. ABIN2868164, Supplier: Log in to see
Application
DNA Isolation (DNAIso)
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Purpose Isolation and purification of circulating and Exosome-associated genomic DNA from urine and cell media
Brand ExoDNAUC™
Sample Type Urine, Cell Culture Supernatant
Characteristics ExoDNAUC™ Kit (isolation and purification of circulating and Exosome-associated genomic DNA) combines the ability of ExoPure™ to isolate exosomes from urine and cell media with a user friendly system of DNA purification. Isolated exosomes are lysed with the appropriate lysis buffer and exosome DNA is purified by and exosome DNA is purified by spin columns and optimized buffers with a fast turnaround time. In addition, ExoDNAUC™ provides lyophilized exosomes to be used as quality controls for exosome capture and DNA extraction. The procedure takes about 1 hour and 45 minutes for yielding purified genomic DNA. Furthermore, ExoDNAUCTM Kit provides lyophilized exosomes to be used as quality controls for exosome capture and DNA extraction. Exosome standards for positive control are also included in the kit. All our kits guarantee high efficiency isolation of circulating and Exosome-associated genomic DNA than similar products.
Components ExoPureTM, Lysis buffer, Proteinase K, Washing buffer 1, Washing buffer 2, Elution buffer, Columns, RNase free elution tubes (1.5 ml), Lyophilized Exosome Standard (Urine/Cell media)
Material not included
  • Single-use and/or pipettes with disposable tips 2-100 μL
  • Pipettes 1 mL and 5 mL for reagent preparation
  • PBS
  • Disposable pipetting reservoirs
  • Ethanol 96 %
Comment

Further details regarding sample type: human urine, cell media

Protocol
    ExoDNAUC™ Assay Protocol:
  1. Urine sample preparation: Centrifuge 10 min at 350 g at RT to eliminate cells and protein aggregates. Save the supernatant.
  2. Cell supernatant sample preparation:
    • 10 min at 300g (save supernatant, discard pellet).
    • 20 min at 1600g (save supernatant, discard pellet).
    • 30 min at 10,000g (save supernatant, discard pellet).
  3. Reagent preparation:
    • Washing Buffer1 and Washing buffer 2: Add the volume of pure ethanol (96 % ) indicated on the label of the bottles of both Washing Buffers.
    • ExoPureTM, Elution buffer and Lysis buffer are ready to use.
  4. Lyophilized Exosome Standards:
    • Reconstitute lyophilized exosome standard by adding 100 μL of deionized water and pipetting the solution up and down 10-15 times, avoiding bubbles. Vortex the reconstituted standard for 60 sec. Briefly centrifuge the tubes containing the standard to ensure that the solution is collected at the bottom of the tube.
  5. Exosome isolation from Urine:
    • Add 1 mL of ExoPureTM reagent to 4 mL of precleared urine.
    • Mix well by pipetting and inverting tube.
    • Incubate on ice for 1 hr.
    • Centrifuge 20 min at 10,000g (centrifuge can be performed at 4 °C or at RT)
    • Discard the supernatant.
    • Centrifuge for 2 min at 1500g to eliminate entirely the supernatant.
    • Resuspend isolated exosomes in 100 μL of 1X PBS.
  6. Exosome isolation from Cell Media:
    • Add 1 mL of ExoPureTM reagent to 1 mL of precleared cell medium.
    • Mix well by pipetting and inverting tube.
    • Incubate on ice for 1 hr.
    • Centrifuge 20 min at 10,000g (centrifuge can be performed at 4 °C or at RT).
    • Discard the supernatant.t
    • Centrifuge for 2 min at 1500g to eliminate entirely the supernatant.
    • Resuspend the pellet in 100 μL of 1X PBS.
  7. DNA Extraction:
    • Lysis:
      1. Add 20 μL of Proteinase K (600 mAU/mL).
      2. Add 200 μL of Lysis Buffer.
      3. Add 5 μL of Poly A Carrier (optional)*.
      4. Mix well by vortexing 30 sec.
      5. Incubate samples at 56 °C for 10 min. * The use of Poly A Carrier results in an increase in DNA yield, in particular when small amounts of DNA are purified through silica membrane columns. No adverse effect on downstream applications such as PCR have been observed (Shaw et al. 2009).
    • DNA Purification:
      1. Add 200 μL of Ethanol 96 % and mix by inverting the tube 6-8 times.
      2. Transfer the mixture in a Spin Column and centrifuge at 10,000 g for 1 min. Discard the flow-through.
      3. Add 500 μL of Washing Buffer 1, centrifuge for 1 min and discard the flow-through.
      4. Add 500 μL of Washing Buffer 2, centrifuge for 1 min and discard the flow-through.
      5. Centrifuge 2 additional min at 16,000g.
      6. Transfer the spin column to an Elution Tube.
      7. Elute the DNA from the column adding 50 μL of Elution Buffer.
      8. Incubate for 5 min at room temperature.
      9. Centrifuge 1 min at 200g.
      10. Centrifuge 1 min at 16,000g.
  8. Sensitivity: ExoDNAUCTM Kit guarantees high efficiency isolation of circulating and Exosome-associated DNA.
Reagent Preparation
  • The RNase free column and elution tubes should be stored at room temperature as well as at 4 °C.
  • The remaining reconstituted Exosome standard stock solution should be aliquoted into polypropylene vials (preferably low binding) and stored at -20 °C for up to one month or at -80 °C for up to six months. Strictly avoid repeated freeze and thaw cycles.
  • All the opened buffers and diluted reagents including ExoPureTM, lysis buffer, washing buffers, ploy A carrier, proteinase K and the elution buffer should be stored at 4 °C.
Restrictions For Research Use only
Storage 4 °C
Storage Comment All the reagents are shipped and stored at +4°C for up to 12 months, if unopened. Briefly centrifuge small vials prior to opening. DO NOT FREEZE!
Expiry Date 12 months