S4530 | EX-WAX™ Paraffin-embedded DNA Extraction Kit

S4530
20 assays  
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      Overview

      Replacement Information
      Description
      Catalogue NumberS4530
      Brand Family Chemicon®
      Trade Name
      • EX-WAX
      • Chemicon
      DescriptionEX-WAX™ Paraffin-embedded DNA Extraction Kit
      OverviewCHEMICON's EX-WAX™ DNA Extraction Kit is intended to extract DNA from paraffin-embedded tissue fixed in 10% Formalin or non-crosslinking fixatives. DNA extracted by this kit is suitable for amplification by PCR.



      Limitations:

      The quality of extracted DNA is directly related to the quality of the embedded tissue. Harsh or extended fixation will adversely affect the results.



      Summary and Principle

      The EX-WAX™ DNA Extraction Kit is qualified on paraffin-embedded mammalian tissues fixed in 10% Formalin or non-crosslinking fixative. DNA is made accessible by protein digestion. DNA is solubilized, while digested proteins are "salted out" and spun to the bottom of the tube. DNA is precipitated, dried under vacuum, and resuspended. Once in solution, the DNA is suitable for amplification by PCR.

      Warnings and Precautions

      1. Wear gloves when isolating and handling DNA to minimize the activity of endogenous nucleases. Use autoclaved pipette tips and 1.5 ml microcentrifuge tubes for additional protection against nucleases.

      2. Volumes are optimized for five 5 μm thick sections.

      3. Qualified on tissue fixed in 10% Formalin or non-crosslinking fixative.

      4. Not recommended for use with fresh, frozen tissues.
      Materials Required but Not Delivered1. Ice cold and room temperature 100% ethanol

      2. Micropipettor and tips (1 to 200 μl)

      3. Vacuum concentrator system (optional)

      4. Microcentrifuge (12,000 rpm)

      5. Freezer (-20°C)

      6. Water bath at 50°C

      7. 1.5 ml microcentrifuge tubes
      References
      Product Information
      Components
      • Sufficient reagents to perform 20 DNA extractions containing three to five 5 μm thick tissue sections per extraction.
      • 90442 Sterile Distilled Water (clear) 1.25 ml Room temp.
      • 90443 Digestion Solution 3.0 ml Room temp.
      • 90444 Extraction Solution 2.0 ml Room temp.
      • 90445 Precipitation Solution 3.0 ml Room temp.
      • 90446 Resuspension Solution 2.0 ml Room temp.
      • 90447 Protein Digesting 25 mg -15 to -25°C
      • Enzyme (Powder)
      Applications
      Key Applications
      • DNA Extraction
      Application NotesPreparation of Reagents

      Protein Digesting Enzyme Solution

      Add 1.25 ml of Sterile Distilled Water (supplied) to the vial of Protein Digesting Enzyme powder. Mix thoroughly. Store unused solution at -15°C to -25°C.



      Extraction Procedure

      1. Cut the tissue sections 5 μm thick. Use 3-5 sections for each extraction.

      2. Cut away excess paraffin and place sections in a 1.5 ml tube. Spin briefly to pellet sections.

      3. Add 1 ml of fresh 100% ethanol (room temperature) and gently vortex for 15 seconds.

      4. Spin for 3 minutes in a microcentrifuge at 12,000 rpm.

      5. Remove ethanol and dry pellet in a vacuum concentrator or at 60°C for 10 minutes with the cap open. If residual ethanol is still present, dry for another 10 minutes.

      6. Add 150 μl of Digestion Solution and 50 μl of Protein Digesting Enzyme Solution to the tube and mix by stirring. Use the end of the micropipettor tip and physically mix the pellet into the solution. Do not pipette up and down (pellet will not dissolve).

      7. Incubate for 4 hours to overnight at 50°C.

      8. Add 100 μl of Extraction Solution and mix by inversion (at least 3 times) for 15 seconds. Over-mixing will excessively shear the DNA.

      9. Spin in a microcentrifuge for 10 minutes at 12,000 rpm. Remove the supernatant and place it in a fresh 1.5 ml tube. This is accomplished by carefully poking the pipette tip through the paraffin layer on top and withdrawing the supernatant, leaving the paraffin and the pellet behind (see Figure 1). If small amounts of paraffin are removed with the supernatant, they will not affect the extraction.

      10. Add 150 μl of Precipitation Solution to the supernatant, invert the tube 3 times, then add 900 μl of ice cold (-20°C) 100% ethanol. Cap the tube and invert several times to mix.

      11. Place at -20°C for at least 1 hour.

      12. Spin in a microcentrifuge for 10 minutes at 12,000 rpm. Discard the supernatant.

      13. Dry the pellet in a vacuum concentrator or at 60°C with the cap open for 10 minutes. If residual ethanol is still present, dry for another 10 minutes.

      14. Add 50 μl of Resuspension Solution and incubate for 1 hour at 50°C in a water bath.

      15. Use 1 μl of resuspended DNA for each PCR reaction and run appropriate PCR controls.



      Troubleshooting

      1. When resuspending the DNA in Resuspension Solution, do not heat the DNA higher than 55°C. This will cause degradation of the DNA.

      2. If the PCR reaction fails, check for the presence and quality of the DNA as follows: Run 10 μl of extracted sample DNA and molecular weight markers on a 0.8% agarose minigel using a 3 mm (width) toothed comb. Run the bromophenol blue dye front approximately 5 cm into the gel. If no sample DNA is observed, try adding 10-40 μl of sample DNA to the PCR reaction or extract a greater number of tissue sections. If the amount of tissue in the block is small, extract more sections, but if the amount of tissue is very large, you may want to try less.
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Packaging Information
      Material Size20 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      SDS

      Title

      Safety Data Sheet (SDS) 

      Certificates of Analysis

      TitleLot Number
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2119196 2119196
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2135699 2135699
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 1963113 1963113
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 1973416 1973416
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 1990786 1990786
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2021768 2021768
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2032313 2032313
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2034716 2034716
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2052930 2052930
      EX-WAX DNA Extraction Kit (for Paraffin-Embedded Tissue) - 2194183 2194183

      References

      Reference overviewPub Med ID
      Phase I/IIa Study of Cilengitide and Temozolomide With Concomitant Radiotherapy Followed by Cilengitide and Temozolomide Maintenance Therapy in Patients With Newly Diagnosed Glioblastoma.
      Stupp R, Hegi ME, Neyns B, Goldbrunner R, Schlegel U, Clement PM, Grabenbauer GG, Ochsenbein AF, Simon M, Dietrich PY, Pietsch T, Hicking C, Tonn JC, Diserens AC, Pica A, Hermisson M, Krueger S, Picard M, Weller M
      J Clin Oncol  2010

      Show Abstract
      20439646 20439646
      Evaluation status and prognostic significance of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in pediatric high grade gliomas.
      Francesca R Buttarelli,Maura Massimino,Manila Antonelli,Libero Lauriola,Paolo Nozza,Vittoria Donofrio,Antonella Arcella,Maria A Oliva,Concezio Di Rocco,Felice Giangaspero
      Child's nervous system : ChNS : official journal of the International Society for Pediatric Neurosurgery  26  2010

      Show Abstract
      20552207 20552207
      Treatment of recurrent malignant gliomas with fotemustine monotherapy: impact of dose and correlation with MGMT promoter methylation.
      Alessandra Fabi,Giulio Metro,Michelangelo Russillo,Antonello Vidiri,Carmine Maria Carapella,Marta Maschio,Francesco Cognetti,Bruno Jandolo,Maria Alessandra Mirri,Isabella Sperduti,Stefano Telera,Mariantonia Carosi,Andrea Pace
      BMC cancer  9  2009

      Show Abstract Full Text Article
      19335893 19335893
      Frequency of bcl-2 gene rearrangement in B-cell Non-Hodgkin's lymphoma.
      Adeel Arif, Shahid Jamal, Sajid Mushtaq, Suhaib Ahmed, Azhar Mubarik
      Asian Pacific journal of cancer prevention : APJCP  10  237-40  2009

      Show Abstract
      19537891 19537891
      Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma.
      Ilse Vlassenbroeck, Stéphane Califice, Annie-Claire Diserens, Eugenia Migliavacca, Josef Straub, Ivano Di Stefano, Fabrice Moreau, Marie-France Hamou, Isabelle Renard, Mauro Delorenzi, Bruno Flamion, James DiGuiseppi, Katja Bierau, Monika E Hegi
      The Journal of molecular diagnostics : JMD  10  332-7  2008

      Show Abstract Full Text Article
      18556773 18556773
      MGMT gene silencing and benefit from temozolomide in glioblastoma.
      Monika E Hegi, Annie-Claire Diserens, Thierry Gorlia, Marie-France Hamou, Nicolas de Tribolet, Michael Weller, Johan M Kros, Johannes A Hainfellner, Warren Mason, Luigi Mariani, Jacoline E C Bromberg, Peter Hau, René O Mirimanoff, J Gregory Cairncross, Robert C Janzer, Roger Stupp
      The New England journal of medicine  352  997-1003  2005

      Show Abstract
      15758010 15758010