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AM41 Suicide Track™ DNA Ladder Isolation Kit

Suicide Track™ DNA Ladder Isolation Kit: Ficha de datos de seguridad (MSDS o SDS), certificado de análisis y de calidad (CoA y CoQ), expedientes, folletos y otros documentos disponibles.
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      Descripción

      Replacement Information
      Description
      OverviewTwo optimized protocols are included to allow for the visualization of either DNA ladder fragments only or both fragments and intact DNA.
      Catalogue NumberAM41
      Brand Family Calbiochem®
      Materials Required but Not Delivered• 2-Propanol (Isopropyl alcohol)
      • Ethanol, 100% and 70%
      • *Tris Base
      • *0.5M EDTA, pH 8.0
      • *Glacial acetic acid
      • Nucleic acid dye (such as SYBR® Green I 10,000 X solution or 5 mg/ml Ethidium Bromide 10,000X solution)
      • Agarose
      • Microcentrifuge tubes
      • Microcentrifuge
      • Water bath capable of incubation at 37°C and 50°C
      • Horizontal gel electrophoresis apparatus
      • 2-20 µl, 20-200 µl, and 200-1000 µl precision pipetters with disposable tips

      *Use these reagents to prepare 1 liter of 50X TAE (Tris-Acetate-EDTA) Buffer as follows:
      Dissolve 242 g Tris base in 750 ml of dH2O. Add 100 ml of 0.5 M EDTA and 57.1 ml of glacial acetic acid. Adjust final volume to 1 liter with dH2O.
      References
      ReferencesEarnshaw, W. C. 1995. Current Opinion in Cell Biology 7, 337.
      Miller,T.E., et al. 1994. In Apoptosis II: The Molecular Basis of Apoptosis in Disease, Current Communications in Cellular and Molecular Biology 8, 357.
      Miller, T.E., 1993. BioTechniques 15, 1042.
      Schwartzman, R. A., and Cidlowski, J. A. 1993. Endocrine Reviews 14, 133.
      Fawthrop, D.J., et al. 1991. Arch. Toxicol. 65, 437.
      Wyllie, A.H. 1980. Nature 284, 555.
      Wyllie A.H., et al. 1980. Int. Rev. Cytol. 68, 251.
      Kerr, J.F.R., et al. 1972. Br. J. Cancer 26, 239.
      Product Information
      Detection methodEthidium bromide
      Form25 Extractions
      FormatAgarose gel electrophoresis
      Kit containsExtraction Buffer, Lysis Buffer, RNase Solution, DNA Isolation Solution, Pellet Paint® Co-Precipitant (Cat. No. 69049-3), DNA Ladder Control, Loading Buffer, DNA Markers, Resuspension Buffer, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time27 h
      Sample TypeAdherent or suspension cells
      Species Reactivity
      • A Broad Range Of Species
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20-36/37/38-41-42/43

      Harmful by inhalation.
      Irritating to eyes, respiratory system and skin.
      Risk of serious damage to eyes.
      May cause sensitization by inhalation and skin contact.
      S PhraseS: 26-36

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      Product Usage Statements
      Intended useThe Calbiochem® Suicide-Track™ DNA Ladder Isolation Kit is an optimized system for the purification and visualization of DNA fragments from apoptotic cells.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsSuicide-Track™ kit components are shipped on dry ice. Upon receipt, store kit at -20°C in a non-frostfree freezer. Once kit components have been thawed for use Solution #1, Sodium Acetate, Extraction Buffer, and Resuspension Buffer can be kept at room temperature and the DNA Markers and Gel Loading Buffer can be stored at 4°C. Re-freezing these components, however, does not appear to affect their performance. To avoid reagent loss in tube caps, briefly pulse spin all thawed solutions before removing caps. See Precautions and Recommendations.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsExtraction Buffer, Lysis Buffer, RNase Solution, DNA Isolation Solution, Pellet Paint® Co-Precipitant (Cat. No. 69049-3), DNA Ladder Control, Loading Buffer, DNA Markers, Resuspension Buffer, and a user protocol.
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      AM41 0

      Documentation

      Licencias necesarias

      Título
      PRODUCTO REGULADO POR LA SECRETARÍA DE SALUD

      Suicide Track™ DNA Ladder Isolation Kit Certificados de análisis

      CargoNúmero de lote
      AM41

      Referencias bibliográficas

      Visión general referencias
      Earnshaw, W. C. 1995. Current Opinion in Cell Biology 7, 337.
      Miller,T.E., et al. 1994. In Apoptosis II: The Molecular Basis of Apoptosis in Disease, Current Communications in Cellular and Molecular Biology 8, 357.
      Miller, T.E., 1993. BioTechniques 15, 1042.
      Schwartzman, R. A., and Cidlowski, J. A. 1993. Endocrine Reviews 14, 133.
      Fawthrop, D.J., et al. 1991. Arch. Toxicol. 65, 437.
      Wyllie, A.H. 1980. Nature 284, 555.
      Wyllie A.H., et al. 1980. Int. Rev. Cytol. 68, 251.
      Kerr, J.F.R., et al. 1972. Br. J. Cancer 26, 239.

      Folleto

      Cargo
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protocolo de usuario

      Revision05-October-2011 RFH
      Form25 Extractions
      FormatAgarose gel electrophoresis
      Detection methodEthidium bromide
      Speciesa broad range of species
      StorageSuicide-Track™ kit components are shipped on dry ice. Upon receipt, store kit at -20°C in a non-frostfree freezer. Once kit components have been thawed for use Solution #1, Sodium Acetate, Extraction Buffer, and Resuspension Buffer can be kept at room temperature and the DNA Markers and Gel Loading Buffer can be stored at 4°C. Re-freezing these components, however, does not appear to affect their performance. To avoid reagent loss in tube caps, briefly pulse spin all thawed solutions before removing caps. See Precautions and Recommendations.
      Intended useThe Calbiochem® Suicide-Track™ DNA Ladder Isolation Kit is an optimized system for the purification and visualization of DNA fragments from apoptotic cells.
      BackgroundApoptosis and necrosis are two major mechanisms of cell death. Necrotic cell death occurs due to noxious injury or trauma, while apoptosis is an endogenous cellular process where an external signal activates a metabolic pathway that results in cell death. This form of cell death is a common feature of cellular differentiation and other biological processes that regulate cell number. In addition apoptosis is triggered by various anti-neoplastic agents as well as after the removal of essential growth factors. Apoptosis has been shown to be a highly organized and compartmentalized mode of cell death in contrast to the erratic and lytic process of necrosis. Apoptosis is characterized by chromatin condensation and degradation, a reduction in cell volume, membrane blebbing and solubilization of the nuclear matrix. Pioneering work in the field of apoptosis research demonstrated a correlation between two indicators of this death process (Kerr, et al., 1972). It was shown that following glucocorticoid treatment of rat thymocytes, cells underwent widespread morphological changes (chromatin condensation and cytoplasmic blebbing) that were associated with the incidence of nucleosome excision from chromatin. Nucleosomal excision is caused by endonuclease-mediated digestion of the exposed DNA linker regions between nucleosomes in chromatin. Since the 180-200 base pairs of DNA around a histone core are conformationally protected from digestion, this endonuclease-mediated nucleosome excision is observable as a DNA ladder (multimers of approximately 180-200 base pairs) in agarose gels. This characteristic form of DNA degradation is known as a major biochemical hallmark of apoptosis (Wyllie, 1980). This Suicide-Track™ DNA Ladder Isolation Kit is sufficiently sensitive to demonstrate laddering, even when cleaved DNA is present at modest levels.
      Principles of the assayThe Suicide-Track™ DNA Ladder Isolation Kit provides a fast, nonisotopic method for the detection of DNA laddering in either suspension cells or cell monolayers. Two different procedures can be performed with the reagents supplied. Suicide-Track™ components have been optimized to separate apoptotic DNA from high molecular weight, intact, genomic DNA (Procedure 1) or both fragmented DNA and high molecular weight DNA can be recovered from a cell sample (Procedure 2). Both methods involve the rapid lysis of cell samples and inactivation of nucleases. Pellet Paint® Co-Precipitant improves the speed, efficiency, and reliability of nucleic acid precipitation since 1) Pellet Paint® is fluorescently labeled, aiding in the visualization of the DNA pellet, 2) incubation time with Pellet Paint® is only 2 minutes at room temperature, 3) small amounts of nucleic acid can be recovered, and 4) extraction with phenol, chloroform, or other organics is not necessary. Following resuspension in the buffer provided, DNA ladder fragments are separated by standard agarose gel electrophoresis and stained with ethidium bromide or SYBR® Green I to produce high resolution DNA ladders. DNA Markers of appropriate size range have been included for ease of photography and interpretation.
      Materials providedThe Suicide-Track™ kit supplies sufficient reagents for 25 samples.

      • Extraction Buffer (Kit Component No. JA1837-15ML): A buffer used for separation of apoptotic DNA fragments from high molecular weight DNA.
      • Solution #1 (Kit Component No. JA1825-1.38ML): A buffer designed for the rapid lysis of cell samples and the inactivation of nucleases.
      • Solution #2 (Kit Component No. JA1826-.5ML): A solution used for degradation of RNA in the cell lysate.
      • Solution #3 (Kit Component No. JA1827-.625ML): A solution that aids in the isolation of DNA from the cell lysate.
      • 3M Sodium Acetate, pH 5.2 (Kit Component No. JA1828-1.5ML):
      • Pellet Paint® Co-Precipitant (Kit Component No. JA1836-.05ML): A fluorescent nucleic acid carrier designed for alcohol precipitation.
      • Positive Control Cell Pellet (formerly DNA Ladder Control) (Kit Component No. JA1829-1EA): Two vials, each containing a pellet of 1 x 10⁶ HL60 cells treated with 0.5 mg/ml Actinomycin D for 19 h.
      • 6X Gel Loading Buffer (Kit Component No. JA1830-.2ML): A gel loading buffer containing two tracking dyes that do not interfere with the visibility of ethidium bromide stained bands.
      • DNA Markers (Kit Component No. JA1831-.02ML): A mixture of eight double stranded DNA fragments of 2000, 1500, 1000, 750, 500, 300, 150, and 50 base pairs supplied in gel loading buffer.
      • Resuspension Buffer (Kit Component No. JA1832-14ML): 10 mM Tris pH 7.5, 1 mM EDTA
      Materials Required but not provided• 2-Propanol (Isopropyl alcohol)
      • Ethanol, 100% and 70%
      • *Tris Base
      • *0.5M EDTA, pH 8.0
      • *Glacial acetic acid
      • Nucleic acid dye (such as SYBR® Green I 10,000 X solution or 5 mg/ml Ethidium Bromide 10,000X solution)
      • Agarose
      • Microcentrifuge tubes
      • Microcentrifuge
      • Water bath capable of incubation at 37°C and 50°C
      • Horizontal gel electrophoresis apparatus
      • 2-20 µl, 20-200 µl, and 200-1000 µl precision pipetters with disposable tips

      *Use these reagents to prepare 1 liter of 50X TAE (Tris-Acetate-EDTA) Buffer as follows:
      Dissolve 242 g Tris base in 750 ml of dH2O. Add 100 ml of 0.5 M EDTA and 57.1 ml of glacial acetic acid. Adjust final volume to 1 liter with dH2O.
      Precautions and recommendations1. For optimal results please read kit instructions prior to use.
      2. To avoid reagent loss in tube caps, briefly pulse spin all thawed solutions before removing caps.
      3. Suicide-Track™ components, with the exception of Solution #1, Sodium Acetate, Extraction Buffer, and Resuspension Buffer, should be kept on ice during use and promptly returned to -20°C. The DNA Markers and Gel Loading Buffer can be stored at 4°C.
      4. Gloves, lab coat, and protective eyewear should be worn.
      5. The DNA Ladder Control is a positive control sample of HL-60 cells induced to undergo apoptosis with Actinomycin D. It should be processed in the same manner as experimental cell samples beginning with either DNA Extraction Protocol Procedure 1 or Procedure 2.
      6. Pellet-Paint® Co-Precipitant is compatible with both isopropanol and ethanol precipitation. Therefore, 1.4 ml of 100% ethanol may be substituted for 2-Propanol in the DNA Extraction Protocol. If using ethanol precipitation it will be necessary to split the resultant volume into two tubes. Only 2 ml of Pellet-Paint® should be used regardless of the sample volume.
      7. Isolation of DNA from non-apoptotic cells may prove to be difficult since intact genomic DNA can be viscous and may be hard to tightly pellet. Use less than the recommended amount of cells for DNA isolation from control or untreated samples. Avoid repeated or vigorous pipetting, as these actions may shear the high molecular weight DNA. To prevent contamination of DNA ladder fragments with high molecular weight DNA use Procedure 1. To isolate both intact and fragmented DNA use Procedure 2.
      8. Successful detection of apoptosis by DNA laddering is dependent upon several factors including cell type and DNA content, method (or agent) inducing apoptosis, length of treatment, as well as degree of mechanical manipulation. It may be necessary to optimize these procedures for your particular application. In some experimental models apoptotic DNA fragmentation may be undetectable by DNA laddering or may be masked by necrotic or intact DNA.
      9. There are several points in the Suicide-Track™ procedure where processing can be stopped for convenience. Samples can be stored at -20°C after pelleting the cell sample (following Sample Preparation), after treatment with Solution #3 (following DNA Extraction), or following the final resuspension (DNA Precipitation).
      PreparationSuspension Cells 1. Transfer cell suspension (5 x 105 to 1 x 106 cells) to a microcentrifuge tube. 2. Centrifuge at 1000 x g for 5 min at room temperature. Remove supernatant and continue with DNA Extraction Procedure 1 to selectively extract apoptotic DNA from intact chromatin OR DNA Extraction Procedure 2 to isolate both fragmented and high molecular weight DNA. •At this point samples may be stored at -20°C for Procedure 2 and following Step 4 in Procedure 1 DNA Extraction. Adherent Cells 1. Remove and save media. Process the media sample following the Suspension Cells Protocol. • This media may contain apoptotic cells that have floated loose from the monolayer. Keep this sample separate from the monolayer sample to allow for better separation of apoptotic DNA fragments from intact chromatin. 2. DNA may be extracted from the monolayer using either DNA Extraction procedure. Add 500 µl of Extraction Buffer (Procedure 1) or 55 µl of Solution # 1 (Procedure 2) directly to the monolayer (5 x 105 to 1 x 106 cells) and gently rock dish to cover cells. Gently transfer the cell lysate to a microfuge tube with minimal pipetting. Continue with the appropriate DNA Extraction Protocol as described. • For larger monolayers maintain this ratio of Extraction Buffer or Solution #1 to cells and likewise increase volumes of Solutions #2 and #3. Following incubation with Solution #3 make 100 µl aliquots of the lysate. Aliquots may be stored at -20°C. DNA Extraction • Two protocols are provided: Total DNA can be isolated using Procedure 2 or DNA ladder fragments can be selectively extracted using Procedure 1. • Volumes are for one sample of 5 x 105 to 1 x 106 cells.
      Detailed protocolProcedure 1: Separation of Apoptotic DNA from High Molecular Weight Chromatin

      1. Resuspend the cell pellet in 500 µl of Extraction Buffer.
      2. Incubate for 30 min on ice.
      3. Centrifuge at 15,000-16,000 x g for 5 min at room temperature.
      4. Carefully remove supernatant and transfer to a clean tube. Discard the tube containing HMW chromatin.
      5. Add 20 µl of Solution #2.
      6. Incubate at 37°C for 60 min.
      7. Add 25 µl of Solution #3 and mix gently.
      8. Incubate at 50°C for 1 h to overnight. Continue with the DNA Precipitation Protocol.

      Procedure 2: Isolation of Total DNA - No Separation of Apoptotic DNA from High Molecular Weight Chromatin

      1. Gently resuspend the cell pellet in 55 µl of Solution #1.
      2. Add 20 µl of Solution #2.
      3. Incubate at 37°C for 1 h.
      4. Add 25 µl of Solution #3 and mix gently.
      5. Incubate at 50°C for 1 h to overnight.
      6. Add 500 µl of Resuspension Buffer and mix. Continue with the DNA Precipitation Protocol.

      DNA Precipitation
      1. Add 2 µl of Pellet Paint® Co-Precipitant.
      2. Add 60 µl of 3M Sodium Acetate, pH 5.2. Mix briefly.
      3. Add 662 µl of 2-Propanol.
      4. Mix by inversion and incubate at room temperature for 2 min.
      5. Centrifuge at 15,000-16,000 x g for 5 min. A pink pellet should be visible in the bottom of the tube.
      • In samples that do not contain large amounts of fragmented DNA UV light may be necessary to see this pellet.
      6. Remove the supernatant with a pipet tip. (Do not pour-off).
      7. Rinse the pellet with 500 µl of 70% ethanol. Repeat step 5.
      8. Remove the supernatant with a pipet tip.
      9. Rinse the pellet with 500 µl of 100% ethanol. Repeat step 5.
      10. Remove the supernatant with a pipet tip.
      11. Air dry by placing the inverted tube open on the benchtop for a few min at room temperature.
      12. Resuspend pellet in 50 µl of Resuspension Buffer.

      DNA Gel Electrophoresis
      1. Dilute 50X TAE (see page 4) to 1X with dH2O.
      2. In a glass container prepare enough 1.5% agarose in 1X TAE to pour a gel ~0.75 cm thick.
      3. Heat the solution in a boiling water bath or microwave oven until the agarose is totally dissolved. Cool the solution to 60°C.
      4. Pour molten agarose into a prepared electrophoresis chamber and position a gel comb to form sample wells.
      5. After the gel is completely solidified remove the comb and position the gel in the gel buffer tank.
      6. Add enough 1X TAE to cover the gel to a depth of 1-2 mm.
      7. Pulse-spin processed DNA ladder sample to bring down any condensation in tube.
      8. Transfer 21 µl of DNA ladder sample to a clean centrifuge tube.
      9. Add 4 µl of 6X Gel Loading buffer and load sample onto the gel.
      10. Pulse-spin DNA Markers and load 5 µl onto the gel.
      11. Assemble the lid onto the electrophoresis chamber and attach electrical leads so that the DNA will migrate toward the positive (red/anodic) lead.
      12. Run at ~50 constant volts until dye front is 1-2 cm from bottom of gel.

      Detection of DNA Ladders
      1. Dilute 5 µg/ml ethidium bromide to 0.5 mg/ml in 1X TAE Buffer.
      • Alternatively dilute 10,000X SYBR® Green I to 1X in TAE, pH 8, and stain as described. The pH of the TAE must be 8.0. A significant decrease in sensitivity is observed at pH less than 7.5 and greater than 8.3 with SYBR® Green I.
      2. Stain gel for 0.5-1 h in 0.5 mg/ml ethidium bromide.
      • To save time the gel may be stained directly in the electrophoresis chamber during the last hour of electrophoresis.
      3. Visualize stained DNA by UV illumination.
      4. Destain gel in 1X TAE if necessary.
      Assay characteristics and examplesFollowing gel electrophoresis and staining of a 1.5% agarose gel the DNA Markers should be visible as 2000, 1500, 1000, 750, 500, 300, 150, and 50 base pair fragments. The smallest fragment in the DNA ladder should be approximately 180 base pairs. This length of DNA corresponds to the stretch of nucleotides associated with one histone complex, which is therefore protected from apoptotic endonuclease cleavage. Higher bands in a typical DNA ladder represent multiples of this monomeric DNA length. Intact or uncleaved high molecular weight DNA should migrate just below the sample well. Since a small percentage of cells naturally die in culture, it is normal to observe a small amount of DNA laddering from control cell populations. Pellet Paint® is positively charged and fluoresces under UV light. It can sometimes be observed as a wide band above the sample wells since it migrates in the direction of the cathode.

      Figure 1: Detection of Apoptotic DNA in Adherent and Non-Adherent Cell Lines.

      Apoptosis was induced in HL-60 (Lane 1), Jurkat (Lane 2), and SW620 (Lane 3) cells by treatment with 0.5 mg/ml Actinomycin D. DNA was then extracted from each of the cell lines using the Suicide-Track™ DNA Ladder Isolation Kit Procedure 2. Agarose gel electrophoresis was followed by ethidium bromide staining. DNA markers were electrophoresed as a base pair reference (Lane M).


      The Suicide-Track™ DNA Ladder Isolation Kit has been validated at Calbiochem using several in vitro models of apoptosis including UV irradiation and the Actinomycin D-induced death of Jurkat, Daudi, or HL-60 cells. Detection of DNA fragmentation by FragEL™ and Nucleosome ELISA (Cat. No. QIA25) was used to verify the presence of DNA laddering in the UV and Actinomycin D treated cultures.

      Figure 2: Gel

      Agarose gel electrophoresis of DNA isolated from promyelocytic leukemia cells using the Calbiochem® DNA Ladder Isolation Kit. Procedure 2. HL-60 cells were either left untreated (Lane 1) or treated with 0.5 µg/ml Actinomycin D (Lane 2). DNA markers (M) were electrophoresed as a base pair reference (Lane M).


      Figure 3: Stain

      Fluorescein-FragEL™ (Cat. No. QIA39) staining of normal and apoptotic HL-60 cells induced to undergo apoptosis (HL-60) with 0.5 µg/ml Actinomycin D. Cells were fixed and Suicide-Track™ deposited on glass slides using a cytospin. Analysis was performed using fluorescence microscopy.


      Figure 4: Fluorescence

      Fluorescein-FragEL™ DNA Fragmentation Detection Kit (Cat. No. QIA39) staining of normal (Left panel) and HL-60 cells induced to undergo apoptosis with 0.5 µg/ml Actinomycin D (Right panel). Cells were fixed and labeled using the protocol for cells in suspension. Analysis of 5000 cells was performed by flow cytometry.
      Sensitivity Notes

      Figure 5: Suicide-Track™ Sensitivity Study: Percent of apoptotic cells detectable.

      HL-60 cells were treated with 0.5 µg/ml Actinomycin D for 19 h. Apoptotic cells were then mixed with untreated HL-60 cells to achieve a total of 1 x 106 cells (Top) or 5 x 105 cells (Bottom). Total DNA was isolated using Procedure 2 (Top) and DNA ladder fragments were selectively extracted using Procedure 1 (Bottom). Samples were electrophoresed through a 1.5% agarose gel and stained with ethidium bromide as described in the protocol. Arrows denote the migration of Pellet Paint® Co-Precipitant and high molecular weight (HMW) or intact DNA. The intensity of staining in the bottom panel is less because fewer cells were used (see figure below). Ten- percent apoptosis is detectable.

      Figure 6: Suicide-Track™ Sensitivity Study.

      HL-60 cells were treated with Actinomycin D as previously described. DNA ladders were isolated from 1 x 106 cells (Lane 1), 0.5 x 106 cells (Lane 2), or 0.25 x 106 cells (Lane 3) using Procedure 2. Processed samples and DNA Markers were run on a 1.5% agarose and stained with ethidium bromide (Lane M).
      Registered TrademarksCalbiochem® and PelletPaint® are registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ and Suicide Track™ are trademarks of EMD Chemicals, Inc.