PicoPure DNA Extraction Kit

Transcription

1 PicoPure DNA Extraction Kit Prepare PCR-ready DNA Fast Go Direct to PCR The PicoPure DNA Extraction Kit offers an easy, streamlined genomic extraction procedure that produces PCR-ready DNA. Extract and amplify DNA in the same tube, without organic extraction or spin columns. Recover DNA from as few as ten cells procured by Laser Capture Microdissection (LCM) or from milligrams of tissue. The PicoPure DNA Extraction Kit provides conveniently packaged, stable Proteinase K, PCR-compatible DNA Reconstitution Buffer and a complete user guide. In this reformatted Kit, each of ten lyophilized Proteinase K aliquots can be freshly reconstituted to make 150 µg of PicoPure DNA Extraction Solution enough for 150 x 10 µl CapSure HS LCM Cap extractions or 30 x 50 µl CapSure Macro LCM Cap extractions. Maximize DNA Recovery Because DNA is not lost due to organic extractions or spin columns, the PicoPure Kit enables reliable and reproducible DNA recovery from as few as ten cells. Reproducible DNA extraction and PCR amplification of multiple single-copy genes from ten Laser Capture Microdissected cells. Cells were microdissected from human peripheral white blood cell cytospins, and DNA was extracted using 10 µl of PicoPure DNA Extraction Solution. The extracts were analyzed directly in 35-cycle PCRs using primers for three single-copy genes. M: DNA Marker (Sigma). Lane 1-5: 536 bp human β-globin gene fragment. Lanes 6-10: 245 bp cystic fibrosis gene fragment (CFTR). Lanes 11-15: 125 bp human p53 gene fragment. Lane 16: No template, negative control PCR. PCR studies illustrate the advantages of the PicoPure Kit. Equal amounts of DNA were transferred to PCR, either directly in the PicoPure Kit Buffer or after purification using a DNA Extraction Kit from a leading manufacturer. Quantitative Real-time PCR (QRT-PCR) demonstrates that the PicoPure Kit samples amplify earlier, suggesting more DNA present, and amplify more consistently, indicating less sample variation, than columnpurified samples. The PicoPure Kit s reliable high DNA recovery enables increased detection sensitivity, for example, when amplifying single-copy genes from small samples in mutation analysis or genotyping assays. This high DNA recovery makes the Kit ideal for small microdissected samples, such as those prepared by LCM. Superior DNA recovery using the PicoPure DNA Extraction Kit. Five, 1 ng replicates of DNA were diluted in 10 µl PicoPure DNA Extraction Buffer, or using the other manufacturer s kit, five, 1 ng replicates of DNA were column purified and concentrated to 10 µl. A human beta-globin gene fragment was then amplified from the samples by Real-Time PCR (40 cycles). Cycle thresholds for the PicoPure Kit samples were 28.9 ± 0.28 and 32.4 ± 1.35 for the column-purified samples, respectively.

2 Extract DNA for Any PCR Application DNA extracted with the PicoPure Kit is compatible with endpoint PCR performed in the same 0.5 ml tube used for DNA extraction. For gene copy number quantitation, DNA from very small samples can be directly amplified by QRT-PCR without further purification, using platforms such as the LightCyler System (Roche). For highly sensitive mutation and genotype analysis, DNA can be extracted and directly amplified from small samples, and gene fragments can be separated by dhplc (denaturing High-Performance Liquid Chromatography), using platforms such as the WAVE System (Transgenomics) Cells 100 Cells 10 Cells PicoPure DNA extraction and quantitation of Her2/neu gene copy number by Real-Time PCR. 10, 100, and 1000 SKBR3 cells were microdissected from cell smears using LCM. DNA was extracted using 10 µl of PicoPure DNA Extraction Solution and, without further purification, all 10 µl were used in a 20 µl LightCycler reaction. The number of gene copies per sample was determined and divided by the number of cells per sample to determine the number of gene copies per cell. PicoPure DNA Extraction Solution is compatible with endpoint PCR and amplicon detection using dhplc. DNA was extracted from 10 peripheral white blood cells per sample using the PicoPure DNA Extraction Kit. This DNA was used, without further purification, in PCRs to amplify a 125 bp human p53 gene fragment and a 245 bp human cystic fibrosis gene (CFTR) fragment. The PCR products were then separated and detected using the dhplc WAVE System. Data courtesy of Scott Hamlin and Nicolas Neckelmann, Transgenomics, Inc., San Diego, CA. Ordering Information Catalog # KIT0103 Catalog # KIT0204 Description PicoPure DNA Extraction Kit 1500 µl Buffer (150 CapSure HS/30 CapSure Macro LCM Cap extractions) Description PicoPure RNA Isolation Kit, enough for 40 RNA isolations KIT0401 HistoGene LCM Frozen Section Staining Kit, enough for 72 tissue sections KIT0420 HistoGene LCM Immunofluorescence Staining Kit, enough for 32 slides Use with Most Tissue and Cell Preparation Procedures KIT0201 RiboAmp RNA Amplification Kit, enough for 10 amplifications The PicoPure DNA Extraction Kit enables successful recovery of genomic DNA from animal tissue section and cell samples prepared using a wide range of methods 1. Superior results are obtained from paraffin-embedded formalinfixed tissue sections, frozen tissue sections, ethanol fixed cells, and cytological smears. 1 Not recommended for use with whole blood, plant or fungal samples. KIT0215 KIT0208 RiboAmp HS RNA Amplification Kit (5 x 2 round samples) RiboAmp OA RNA Amplification Kit (10 x 2 round samples) KIT0206 RiboAmp OA RNA Amplification Kit (20 x 2 round samples) PicoPure DNA extraction and β-globin gene amplification from LCM samples prepared by various methods. Cell and tissue samples were prepared for LCM using the methods listed below. LCM was used to collect samples. DNA was extracted using the PicoPure DNA Extraction Kit, a single-copy 536 bp human β-globin fragment was amplified from each sample using standard protocols, and the products were electrophoresed on a 10% polyacrlamide/tbe gel and stained with SYBR Gold Nucleic Acid Stain (Molecular Probes). M: DNA marker. Lanes 1-3: ethanol-fixed SKBR3 cells. Lanes 4-6: formalin-fixed, paraffin-embedded breast tissue. Lanes 7-9: frozen, ethanol-fixed foreskin tissue. Lane 10: positive PCR control of 1 ng human genomic DNA template. Lane 11: negative control, no PCR template. KIT0207 KIT0209 LCC1701 LCC1702 LCC1703 RiboAmp OA RNA Amplification Kit (60 x 2 round samples) RiboAmp OA 1 Round RNA Amplification Kit (20 x 1 round samples) Veritas TM Microdissection Instrument (laser capture microdissection (LCM) only) Veritas Microdissection Instrument (LCM and fluorescence) Veritas Microdissection Instrument (LCM and laser cutting) Arcturus Bioscience, Inc. 400 Logue Avenue Mountain View, CA USA tel fax contact@arcturusbiosicence.com RiboAmp, PicoPure, HistoGene, PixCell, CapSure and ExtracSure are trademarks owned by Arcturus. All other trademarks shown are the property of their respective owners. Polymerase Chain Reaction (PCR) is a patented process owned by Hoffman-La Roche AG. For research use only. Not for use in diagnostic procedures. LCC1704 LCM0211 LCM0213 Veritas Microdissection Instrument (LCM, laser cutting and fluorescence) CapSure Macro LCM Caps (plastic carriers) CapSure HS LCM Starter Pack with Alignment Tray and Incubation Block P/N Rev C

3 User Guide Pico Pure DNA Extraction Kit Catalog # KIT0103 Version D For Research Use Only

4 PicoPure TM DNA Extraction Kit

5 Table of Contents I. Introduction A. Background B. Storage and Stability C. Quality Control D. Material Safety and Data Sheets II. Kit Components A. Reagents and Supplies in Kit III. Equipment and Materials A. Equipment and Materials IV. Protocol A. Overview B. Time Requirements C. Before You Begin D. DNA Extraction Protocol: Cell Pellet and Tissue Section Samples E. DNA Extraction Protocol: CapSure HS LCM Cap Samples F. DNA Extraction Protocol: CapSure Macro LCM Cap Samples V. Troubleshooting A. Incomplete Digestion of Cells off the CapSure Macro LCM Cap B. Buffer Leaks During Incubation C. Unable to Amplify DNA

6 PicoPure TM DNA Extraction Kit Legal Notices Copyright 2002 Arcturus Engineering, Inc. All Rights Reserved. PixCell is a registered trademark, and CapSure, ExtracSure, HistoGene, PicoPure, and RiboAmp are trademarks of Arcturus Engineering, Inc. Other trademarks used in this manual are the property of their respective owners. The PCR process is covered by patents owned by Hoffmann-La Roche Inc. and F. Hoffman-La Roche Ltd. Warranty Arcturus Engineering, Inc. warrants that the products described in this manual meet the performance standards described in literature published by the company. If a product fails to meet these performance standards, Arcturus will replace the product or issue credit for the full purchase price, including delivery charges. Arcturus provides no other warranties of any kind, expressed or implied. Arcturus warranty liability shall not exceed the purchase price of the product and shall not extend to direct, indirect, consequential or incidental damages arising from the use, results of use, or improper use of its products. The PicoPure DNA Extraction Kit is intended for research purposes only and is not intended for diagnostic or other purposes. 2

7 Introduction I. Introduction A. Background The PicoPure DNA Extraction Kit provides a fast and easy genomic DNA extraction procedure. Conveniently packaged reagents for preparing PCR-ready DNA from animal tissue and cell samples prepared using a variety of methods are included. No organic extractions or spin columns are needed, enabling very high DNA recovery from samples and single-tube DNA extraction and subsequent amplification. This streamlined method makes the kit ideal for processing small samples, of as few as ten cells, where maximum DNA recovery is critical. However, the extraction procedure is equally well suited for larger samples of up to several milligrams of tissue or cell pellets. The Kit uses an optimized Proteinase K extraction procedure, and features lyophilized, premium-quality enzyme in convenient aliquots. These are freshly reconstituted before each use for optimal enzymatic activity. DNA extracted using the PicoPure Kit is ready for use in endpoint or quantitative real-time PCR assays. DNA can be extracted from tissue prepared using a wide variety of preparation methods. Superior results can be obtained from paraffin-embedded, formalin-fixed tissue sections, frozen tissue sections, ethanol-fixed cells, and fresh or previously frozen cell pellets. The method is not designed for DNA extraction from whole blood, plant, or fungal samples. A General DNA Extraction Procedure is provided in Section IV.D for processing non-microdissected samples. Procedures are also provided in Sections IV.E and IV.F for processing small samples obtaining using Laser Capture Microdissection (LCM) with Arcturus PixCell LCM System. Separate procedures are provided for using CapSure HS or Macro LCM Caps. The PicoPure DNA Extraction Kit has been used successfully with most tissue specimens, including archived tissue samples. For PCR, the kit has been tested using Taq polymerase from a variety of suppliers. If you intend to use the kit with cells derived from archived tissue samples, or if you have any other questions about the compatibility of the kit with other substrates or materials, please contact Arcturus Technical Services before you begin by calling or , or send an to techsupport@arctur.com. The Kits are tested to ensure the absence of DNA and DNases, and for compliance with other quality measurements. For example, each lot of Proteinase K is reconstituted in Reconstitution Buffer and tested for protease activity. In addition, functional testing is performed according the protocols described in this user guide. 3

8 PicoPure TM DNA Extraction Kit B. Storage and Stability PicoPure DNA Extraction Kit reagents are shipped at room temperature. Upon receipt, the kits should be stored at room temperature in a cool dry place. Proteinase K should be used immediately after reconstitution. Freeze thawing is not recommended and will decrease the stability and shelf life of the enzyme. Reconstitution Buffer contains sodium azide, a potent toxin which may be fatal if swallowed or absorbed through skin. Use appropriate precautions. C. Material Safety and Data Sheet Material Safety and Data Sheets (MSDS) for kit chemical components are available from Arcturus Technical Services or may be downloaded from Call or , or send to techsupport@arctur.com. D. Related Products from Arcturus Arcturus HistoGene LCM Frozen Section Staining Kit, PicoPure RNA Isolation Kits, and RiboAmp RNA Amplification Kits are optimized for use in LCM procedures. Additional kits are under development. For an updated list of available kits, please contact Arcturus Technical Services at or , or send an inquiry to techsupport@arctur.com. You may also visit Arcturus web site at for current product information. Related Products Catalog # LCM0201 LCM0204 LCM0203 LCM0203 Product CapSure Macro LCM Caps CapSure HS LCM Caps CapSure HS LCM Caps Starter Pack Incubation Block for ExtracSure Device 4

9 Kit Components II. Kit Components A. Reagents and Supplies in Kit ThePicoPure DNA Extraction Kit comes with the following items:! Proteinase K 10 x 0.5 ml Vials! Reconstitution Buffer 1 x 2.0 ml Vials The PicoPure DNA Extraction Kit has been optimized for use with the reagents provided. Substitution of other digestion enzymes or buffers may yield unsatisfactory results. 5

10 PicoPure TM DNA Extraction Kit III. Equipment and Materials Equipment and Materials Ensure that you have ready access to the following laboratory equipment and materials before you begin. These items are not included in the PicoPure DNA Extraction Kit: 1. Equipment! Microcentrifuge (capable of 4000 x g with microfuge tube rotor)! Standard laboratory incubator set at 65 C! Heating block for 0.5 ml microcentrifuge tubes (95 C)! Hand-held pipettor! LCM Cap Insertion Tool (provided with the PixCell LCM System) If using CapSure HS LCM Caps:! ExtracSure Sample Extraction Device (Cat. # 0208)! Alignment Tray (Cat. # LCM0504)! Incubation Block (Cat. # LCM0505)! Clean forceps! Gloves 2. Materials! LCM Captured Cells on either CapSure Macro LCM Cap (Cat. # 0201) or CapSure HS LCM Cap (Cat. # 0204)! Nuclease-free pipette tips! Ice in ice bucket! 0.5 ml microcentrifuge tubes (either standard or thin-walled). CapSure Macro LCM Caps, CapSure HS LCM Caps, and the ExtracSure Device are compatible with 0.5 ml autoclaved thin-walled reaction tubes (Applied Biosystems; N ). 6

11 IV. Protocol A. Overview To ensure best results, please take a few minutes to review this protocol before starting and to organize the equipment and materials needed to perform the protocol. The PicoPure DNA Extraction Kit extracts DNA from cells captured using either CapSure Macro LCM Caps or CapSure HS LCM Caps. We recommend using 100 cells as a routine starting sample until amplification conditions are optimized for fewer cells. The PicoPure DNA Extraction Kit can also be used to extract DNA from small sections of tissue and from cultured cells. The PicoPure DNA Extraction Kit includes sufficient material for 30 extractions when using CapSure Macro LCM Caps and 150 extractions when using CapSure HS LCM Caps. B. Time Requirements Total processing time depends on the tissue source. Extraction from non-formalin fixed samples is very efficient and requires only three hours incubation time. Due to the cross linking that occurs during fixation, DNA extraction from formalin fixed tissue requires a minimum of 16 hours incubation time. C. Before You Begin 1. Pre-heat incubator to 65 C. If using CapSure HS Caps with the ExtracSure device, place heating block in incubator so it can be at the proper temperature when samples are ready for extraction. 2. Centrifuge one vial of Proteinase K for 30 seconds at 1000 x g prior to addition of Reconstitution Buffer. 3. Set an ice bucket with ice at your benchtop work area. 7

12 PicoPure TM DNA Extraction Kit Protocol D. DNA Extraction Protocol: Cell Pellet and Tissue Section Samples The following protocols are recommended for extracting DNA from cell pellets or formalin-fixed, paraffin-embedded tissue sections Pipette 155 µl of PicoPure DNA Extraction Buffer into one vial of Proteinase K. Gently vortex the tube to mix the reagents and complete dissolve the reagent. Immediately place the tube on ice and use as soon as possible. Excessive mixing may denature the Proteinase K. Follow a. for cells or b. for non-fixed and fixed tissue, respectively. a. Cells 1. Pellet 1 x x 10 6 cells in a 1.5 ml microcentrifuge tube. 2. Remove the supernatant. 3. Add 150 µl of Proteinase K DNA Extraction Solution to the tube. 4. Vortex gently to mix cells thoroughly. 5. Incubate sample at 65 C for three hours. 6. Place sample tubes at 95 C for ten minutes to inactivate Proteinase K. b. Fixed tissue sections 1. For paraffin-embedded tissue, remove paraffin by immersing the tissue section in approximately 15 ml of freshly poured xylene, two times, each for five minutes. If the tissue was fixed in ethanol, remove the tissue from the ethanol, and air-dry the tissue for five minutes. 2. Carefully scrape µg of tissue into a 1.5 ml microcentrifuge tube. 3. Add 150 µl of Proteinase K DNA Extraction Solution to the tube. 4. Vortex gently to mix thoroughly. 5. Incubate at 65 C for up to 24 hours. 6. Place sample tubes at 95 C for ten minutes to inactivate Proteinase K. 3. Proceed directly to PCR or store digested samples at 20 C. 8

13 E. DNA Extraction Protocol: CapSure HS LCM Cap Samples 1. Pipette 155 µl of Reconstitution Buffer into one vial of Proteinase K. Gently vortex the tube to mix the reagents and immediately place the tube on ice. Completely dissolve reagent, excessive mixing can denature Proteinase K. 2. Assemble the ExtracSure Sample Extraction Device onto the CapSure HS LCM Cap. a. Using clean forceps, remove the cap from the Cap Insertion Tool and place into the Alignment Tray (sample facing up). Make sure that the cap snaps securely and lays flat on the bottom of the opening of the Alignment Tray. Use Proteinase K Extraction solution as quickly as possible following reconstitution. Discard any unused Proteinase K solution. For best results, preheat Incubation Block in 65 C incubator. Forceps CapSure HS LCM Cap Cap Insertion Tool Forceps CapSure HS LCM Cap Alignment Tray b. Position the ExtracSure Device over the cap using clean forceps. The fill-port should be facing up. ExtracSure Sleeve ExtracSure Sample Extraction Device c. Using forceps, push the ExtracSure Device down onto the cap until it snaps securely into place. Use forceps to ensure that the cap is firmly attached to the ExtracSure Device. Forceps ExtracSure Sample Extraction Device 9

14 PicoPure TM DNA Extraction Kit 3. Pipette 10 µl of Proteinase K Extraction Solution into the microchamber formed by the assembled ExtracSure Device and CapSure HS LCM Cap. Pipettor Tip ExtracSure Sample Extraction Device 0.5 ml Microcentrifuge Tube 4. Using gloved hands, place a 0.5 ml thin-walled reaction tube over the ExtracSure device. 5. Cover the assembly in the Alignment Tray with the Incubation Block and incubate it for the correct time period according to the following table: Heating Block Alignment Tray Sample Type Formalin Fixed/Paraffin Embedded Non Formalin Fixed/Paraffin Embedded Frozen Cytospin & Cell Smears Incubation Time (65 o C) > 16 hours 3 hours 3 hours 3 hours CapSure-ExtracSure Assembly with Microcentrifuge Tube 6. After incubation, remove the assembled CapSure HS LCM Cap and ExtractSure Device from the incubator, place the assembly into a microcentrifuge and centrifuge for one minute at 4,000 x g. 7. Separate the CapSure HS LCM Cap and ExtracSure Device. Close the microcentrifuge tube containing the extract and heat to 95 C for ten minutes in a heating block to inactivate the Proteinase K. Cool the sample to room temperature. Some samples may benefit from longer incubation times, or higher reaction concentrations of Proteinase K. See Troubleshooting. For specific advice, please contact Arcturus Technical Services at or , or send to techsupport@arctur.com. 8. Samples are now ready for PCR analysis. Additional purification should not be required. 10

15 Protocol F. DNA Extraction Protocol: CapSure Macro LCM Cap Samples 1. Pipette 155 µl of Reconstitution Buffer into one vial of Proteinase K. Gently vortex the tube to mix the reagents and place the tube on ice immediately. Completely dissolve reagent, excessive mixing may denature Proteinase K. 2. Pipette 50 µl of Proteinase K Extraction Solution into a 0.5 ml microcentrifuge tube. 3. Insert the CapSure Macro LCM Cap with LCM captured cells into the microcentrifuge tube using the LCM Cap Insertion Tool. 4. Invert the microcentrifuge tube with the inserted CapSure Macro LCM Cap and shake down the 50 µl volume of Proteinase K Extraction solution until it completely covers the inside surface of the CapSure Macro LCM Cap. Cap Insertion Tool CapSure LCM Cap Microcentrifuge Tube Use Proteinase K Extraction Solution as quickly as possible following reconstitution. Discard any unused Proteinase K solution. 5. Incubate the inverted tube at 65 C for the correct time period according to the following table: Sample Type Incubation Time (65 o C) Formalin Fixed/Paraffin Embedded Non Formalin Fixed/Paraffin Embedded Frozen Cytospin & Cell Smears > 16 hours 3 hours 3 hours 3 hours 6. After incubation, remove the tubes from the incubator, place them in a microcentrifuge and centrifuge for one minute at 1,000 x g. 7. Remove the CapSure Macro LCM Cap. Close the microcentrifuge tube containing the extract and heat to 95 C for ten minutes in a heating block to inactivate the Proteinase K. Cool the sample to room temperature. 8. Samples are now ready for PCR analysis. Additional purification should not be required. 11

16 PicoPure TM DNA Extraction Kit Troubleshooting V. Troubleshooting A. Incomplete Digestion of Cells off the CapSure Macro LCM Cap 1. Proteinase K may be inactive. Always use freshly reconstituted Proteinase K. Reconstituted aliquots of Proteinase K can lose enzymatic activity during storage. 2. Proteinase K may have become inactive due to enzyme autodigestion during long incubation periods. For samples requiring prolonged incubation, pipette an additional 10 µl of freshly reconstituted Proteinase K Extraction Solution into the reaction chamber following the first 24 hours of incubation. 3. Samples with large amounts of connective tissue may require longer incubations with Proteinase K for complete digestion. 4. Proteinase K is absent from the sample extraction buffer. Be sure to add Reconstitution Buffer to a fresh vial of Proteinase K before use. B. Buffer Leaks During Incubation 1. If buffer leaks from microcentrifuge tube, ensure that CapSure Macro LCM Cap is properly inserted into the microcentrifuge tube. 2. If buffer leaks from ExtracSure Device, ensure that the device is properly assembled onto the CapSure HS LCM Cap. Do not remove assembled device from Alignment Tray before incubation. 12

17 C. Unable to Amplify DNA 1. Template DNA quantity is insufficient. Increase the number of cells in starting sample or the amount of lysate transfered to amplification reaction. 2. Proteinase K was not inactivated. If Proteinase K was not completely inactivated prior to addition of PCR reagents, Proteinase K may digest the Polymerase. Following Proteinase K digestion, heat extracted sample for ten minutes above 95 C to inactivate Proteinase K. 3. PCR parameters are not optimized. Make sure PCR primer sequences are correct for target gene amplification. Verify that PCR primers are present in the proper concentrations for the reaction conditions. Ensure that the annealing temperature has been optimized. 4. PCR inhibitors may be present in sample. PCR inhibitors (heme, melanin, certain stains in high concentration such as hematoxylin) may have carried over from tissue preparation. Purify the DNA sample further before PCR. 5. PCR priming is non-specific. Optimize primer concentration and reaction conditions. 13

18 PicoPure TM DNA Extraction Kit Arcturus Arcturus GmbH Arcturus Ltd. Arcturus SARL 400 Logue Avenue Germany United Kingdom France Mountain View, CA tel [49] (0) tel [44] (158) tel [33] (1) tel USA fax [49] (0) fax [44] (158) fax [33] (1) fax p/n