GENECLEAN Kit For Ancient DNA

Transcription

1 Instruction Manual GENECLEAN Kit For Ancient DNA DNA isolation from museum or ancient specimens for PCR, including samples of bone, preserved tissue, or animal by-products One Call One Source A World of Biotechnology Reagents 100 Preps Storage: C Catalog # Revision # NOV MP Biomedicals Fountain Parkway Solon, OH tel: fax:

2 GENECLEAN Kit For Ancient DNA Dirty Samples? Tiny (or Huge) Samples? Tough Samples? No Problem with FastPrep. part of an MP BIO integrated laboratory solution Sample Prep Higher yields. Consistent quality. Super fast. FastPrep lets you get the most usable DNA, RNA and proteins from even your most difficult samples, fast and easy, every time. FREE fastprep DEMO! Learn more at: 2

3 GENECLEAN Kit For Ancient DNA DNA isolation from museum or ancient specimens for PCR, including samples of bone, preserved tissue, or animal by-products 100 Preps Storage: C Catalog # Revision # NOV 3

4 GENECLEAN Kit For Ancient DNA TABLE OF CONTENTS 1. Introduction to the GENECLEAN Kit for Ancient DNA GENECLEAN Kit for Ancient DNA Kit Components Storage of the GENECLEAN Kit for Ancient DNA Important Considerations Before Use Safety Precautions Protocols...7 6a. Manual Protocol...7 6b. Protocol Using Homogenization Matrix/Tubes...8 6c. Protocol for Use with FastPrep Instruments Appendix Notes Recommended Reference Format for Publication Product Use Limitation & Warranty

5 1. Introduction to the GENECLEAN Kit for Ancient DNA The GENECLEAN Kit for Ancient DNA is designed for isolation of DNA from non-viable tissues or samples which have historical or forensic value including samples of bone, preserved tissue or animal by-products. The reagents are formulated carefully to prevent contamination by contemporary DNA. Each preparation is capable of isolating 20 µg of DNA. The resulting samples are readily amplified by PCR or other methods of amplification. 2. GENECLEAN Kit for Ancient DNA Kit Components (100 preps) Name: Volume: Catalog #: DeHybernation Solution A* 110 ml DeHybernation Solution B 110 ml Dehyb Solution A2 18 ml Ancient DNA GLASSMILK* 35 ml Salton Wash # 1* 60 ml Salton Wash #2 60 ml Ancient DNA Alcohol Wash** 15 ml DNA-free Elution Solution 15 ml SPIN Filters 100 each Catch Tubes 100 each Homogenization Matrix/Tubes 5 each *See Section 5 ** See Section 4.1 5

6 GENECLEAN Kit For Ancient DNA 3. Storage of the GENECLEAN Kit for Ancient DNA The GENECLEAN Kit for Ancient DNA is shipped at ambient temperature. Upon receipt, store at C. Optionally, the kit can be stored at 4 C to extend product shelf life. 4. Important Considerations Before Use 4.1 Preparation of Ancient DNA Alcohol Wash The GENECLEAN Kit for Ancient DNA contains Ancient DNA Alcohol Wash, which is a plastic screw-top bottle containing 15 ml of a concentrated salt wash solution. Before using this buffer, add 135 ml of 100% ethanol and mark on the bottle label the date ethanol was added. Ensure that the bottle is securely closed to prevent evaporation, and store at room temperature. 4.2 DeHybernation Solution Selection and Sample Specificity Two different DeHybernation Solutions are supplied with this kit. DeHyb A is a guanidinebased solution; DeHyb B is an aqueous EDTA-based solution. Please be advised, because of the large diversity of samples and their conditions (i.e. degree of preservation and/or environment) it is difficult to determine which DeHyb solution will be most efficacious in each case. Therefore, preliminary experiments may be necessary. 5. Safety Precautions The Ancient DNA GLASSMILK, DeHybernation Solution A, and Salton Wash #1 contain Guanidine thiocyanate. Use with proper precaution. 6

7 6. Protocol 6a. Manual Protocol Incubation with Proteinase K has been reported to increase yields (See Notes in Section 8). 1. Add mg homogenized or powdered sample to 1 ml DeHybernation Solution* in a nucleic acid-free microcentrifuge tube. 2. Incubate at C between 2 and 12 hours with mixing (longer incubation times may be necessary). 3. Centrifuge sample at high speed for 5 minutes to pellet particulate material. Transfer supernatant to a new nucleic acid-free microcentrifuge tube. Add 300 µl Ancient DNA GLASSMILK. Incubate at room temperature for minutes with mixing. 4. Transfer suspension to a SPIN Filter Tube. Centrifuge at 14,000 x g in a microcentrifuge for 1 minute or until liquid transfers to the Catch Tube. (Empty Catch Tube as needed). 5. Add 0.5 ml Salton Wash #1 and centrifuge at 14,000 x g to clean GLASSMILK-DNA complex. 6. Add 0.5 ml Salton Wash #2 and centrifuge at 14,000 x g to clean GLASSMILK-DNA complex. 7. Add 0.5 ml Ancient DNA Alcohol Wash** and centrifuge to empty filter of Wash Solution. Repeat. 8. Empty Catch Tube and centrifuge for 2 minutes to dry GLASSMILK in the SPIN Filter. 9. Place filter into a DNA-free Elution Catch Tube. Add µl DNA-free Elution Solution. Resuspend pellet by hand or briefly vortex (1-2 seconds, any longer will result in damage to the filter). Centrifuge for 1 minute to transfer eluent to Catch Tube. Optional: Elute a second time. 7

8 GENECLEAN Kit For Ancient DNA 10. Remove SPIN Filter and discard. DNA is ready to use in amplification reaction without further manipulation. Note: There are two different Dehyb solutions supplied with this kit. They differ in composition and each is effective in the isolation of DNA. Because the materials from which the DNA is to be extracted vary so greatly we are not able to recommend one over another. * There is an additional detergent supplied with this kit that can be used in conjunction with DeHyb A. In certain applications this detergent (DeHyb A2) has resulted in improved DNA yields. If you choose to use DeHyb A2, then the amount of DeHyb A should be decreased to 850 ul, and 150 ul of DeHyb A2 must be added just prior to incubation or homogenization (vortexing or processing in the FastPrep Instrument). **Be sure to add 100% Ethanol to the Ancient DNA Alcohol Wash prior to first use. See Section 4.1 for instructions on reconstituting Ancient DNA Alcohol Wash. 6b. Protocol Using Homogenization Matrix/Tubes Incubation with Proteinase K has been reported to increase yields (See Notes in Section 8). 1b. Add mg sample (slightly granular form, whole chunks will not homogenize well) to 1 mi DeHybernation Solution* in the Homogenization Matrix/Tube. 2b. Vortex for 15 minutes at full speed. 3b. Incubate at C between 2 and 12 hours with mixing (longer incubation times may be necessary). 4b. Centrifuge sample at high speed for 5 minutes to pellet particulate material. Transfer supernatant to a new nucleic acid-free tube. Centrifuge again for 3 minutes to remove any remaining particulates. Transfer supernatant to a new nucleic acid-free tube. 8

9 5b. Add 300 µl Ancient DNA GLASSMILK. Incubate at room temperature for minutes with mixing. Continue with Step #4 in Manual Protocol (Section 6a). Note: There are two different DeHyb solutions supplied with this kit. They differ in composition and each is effective in the isolation of DNA. Because the materials from which the DNA is to be extracted vary so greatly we are not able to recommend one over the other. *There is an additional detergent supplied with this kit that can be used in conjunction with DeHyb A. In certain applications this detergent (DeHyb A,) has resulted in improved DNA yields. If you choose to use DeHyb A then the amount of DeHyb A should be decreased to 850 ul, and 150 ul of DeHyb A, must be added just prior to homogenization (vortexing). **Be sure to add 100% Ethanol to the Ancient DNA Alcohol Wash prior to first use. See Section 4.1 for instructions on reconstituting Ancient DNA Alcohol Wash. 6c. Protocol for Use with FastPrep Instruments Incubation with Proteinase K has been reported to increase yields (See Notes in Section 8). 1c. Add mg sample (slightly granular form, whole chunks will not homogenize well) to 1 ml of DNA DeHybernation Solution* in a 2 ml Homogenization Matrix Tube. 2c. Process at a setting of 6 in FastPrep Instrument for 5-45 seconds to homogenize sample. Softer samples require shorter times. 3c. Incubate at C between 2 and 12 hours with mixing (longer incubation times may be necessary). 4c. Centrifuge sample at high speed for 5 minutes to pellet particulate material. Transfer supernatant to a new nucleic acid-free tube. Centrifuge again for 3 minutes to remove any remaining particulates. Transfer supernatant to a new nucleic acid-free tube. 9

10 GENECLEAN Kit For Ancient DNA 5c. Add 300 µi Ancient DNA GLASSMILK. Incubate at room temperature for minutes with mixing. Continue with Step #4 in Manual Protocol (Section 6a). Note: There are two different DeHyb solutions supplied with this kit. They differ in composition and each is effective in the isolation of DNA. Because the materials from which the DNA is to be extracted vary so greatly we are not able to recommend one over the other. *There is an additional detergent supplied with this kit that can be used in conjunction with DeHyb A. In certain applications this detergent (DeHyb A,) has resulted in improved DNA yields. If you choose to use DeHyb A then the amount of DeHyb A should be decreased to 850 ul, and 150 ul of DeHyb A, must be added just prior to incubation or homogenization (vortexing or processing in the FastPrep Instrument). 7. Appendix It may be necessary to wash the DNA/Ancient DNA GLASSMILK pellet to enhance purity of DNA if subsequent reactions are not working properly. Wash with 300 µl of a 1:1 solution of acetone: ethanol prior to the wash step (Section 6a, Step #7) in the Manual Protocol. 8. Notes Due to the chemical denaturants contained in both DeHyb Solutions, a pre-incubation with Proteinase K is suggested. The following protocol was submitted by a trusted customer and validated through experimentation. Note: Starting samples are mg of powder drilled from bone. Overnight Soaking Solution: 5 µl 0.5 M EDTA 200 µl 10% SDS 200 µl 20 mg/ml Proteinase K 10

11 1. Incubate samples with rotation at 37 C for hours. 2. Add 1 µl DeHybernation Solution A to each sample and rotate for 2-4 hours at 60 C. 3. Spin samples in a microcentrifuge to pellet particulate. 4. Transfer supernatant to a clean 15 ml tube and add 1.2 ml Ancient DNA GLASSMILK and 3.0 ml DeHybernation Solution A. 5. Rotate samples for 2 hours at C. 6. Centrifuge samples at 4,000 rpm for 1 minute to pellet DNA/Ancient DNA GLASSMILK conjugate. Discard the supernatant. 7. Add 0.5 ml Salton Wash #1 to resuspend pellet, and then transfer to a SPIN Filter tube. 8. Continue on with Step #6 from the Manual Protocol (Section 6a, Pg 8). 9. Recommended Reference Format for Publications (Specific sample) cells/tissues were processed using the GENECLEAN Kit for Ancient DNA (MP Biomedicals, Santa Ana, CA). 11

12 GENECLEAN Kit For Ancient DNA 10. Product Use Limitation & Warranty The products presented in this instruction manual are for research or manufacturing use only. They are not to be used as drugs or medical devices in order to diagnose, cure, mitigate, treat or prevent diseases in humans or animals, either as part of an accepted course of therapy or in experimental clinical investigation. These products are not to be used as food, food additives or general household items. Purchase of MP Biomedicals products does not grant rights to reproduce, modify, or repackage the products or any derivative thereof to third parties. MP Biomedicals makes no warranty of any kind, expressed or implied, including merchantability or fitness for any particular purpose, except that the products sold will meet our specifications at the time of delivery. Buyer s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited to, at our discretion, no replacement or compensation, product credits, refund of the purchase price of, or the replacement of materials that do not meet our specification. By acceptance of the product, Buyer indemnifies and holds MP Biomedicals harmless against, and assumes all liability for, the consequence of its use or misuse by the Buyer, its employees or others, including, but not limited to, the cost of handling. Said refund or replacement is conditioned on Buyer notifying within thirty (30) days of receipt of product. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material(s). FastDNA, FastRNA, FastPrep, QBiogene, and BIO 101 Systems are registered trademarks of MP Biomedicals, LLC. 12

13 Convenient Lysing Matrix Tubes for Every Need MP Biomedicals guarantees the BEST performance from your FastPrep -24 Instrument when used in combination with FastPrep Lysing Matrix Tubes Lysing Matrix A Each impact-resistant 2 ml tube contains garnet matrix and one 1/4 inch ceramic sphere. Extra 1/4 inch ceramic spheres are packaged separately. Lysing Matrix A tubes have orange caps and are found in the FastDNA and FastDNA SPIN Kits. Lysing Matrix A is used for all sample types except soil for the subsequent isolation of genomic DNA. Lysing Matrix B Each impact-resistant 2 ml tube contains 0.1 mm silica spheres. Lysing Matrix B tubes have blue caps and are found in the FastRNA Pro Blue Kit and FastProtein Blue Matrix. Lysing Matrix B is used for lysis of gram positive and gram negative bacteria. Lysing Matrix C Each impact-resistant 2 ml tube contains 1 mm silica spheres.lysing Matrix C tubes have red caps and are found in the FastRNA Pro Red Kit and FastProtein Red Matrix. Lysing Matrix C is used for lysis of yeast and fungi. Lysing Matrix D Each impact-resistant 2 ml tube contains 1.4 mm ceramic spheres. Lysing Matrix D tubes have green caps and are found in the FastRNA Pro Green Kit for isolation of total RNA from plants and animals. Lysing Matrix E Each impact-resistant 2 ml tube contains 1.4 ceramic spheres, 0.1 mm silica spheres, and one 4 mm glass bead. Lysing Matrix E tubes have purple caps and found in the FastDNA SPIN Kit for Soil and the FastRNA Pro Soil Kits. 13

14 GENECLEAN Kit For Ancient DNA Notes: 14

15 Notes: 15

16 Instruction Manual GENECLEAN Kit For Ancient DNA Revision # NOV Worldwide Ordering and Technical Support United States of America Worldwide Headquarters Tel: Toll Free Tel: Fax: Toll Free Fax: Europe Toll Free Phone: Toll Free Fax: Australia MP Biomedicals Australasia Pty Ltd Tel: Fax: Belgium MP Biomedicals Tel: Fax: France MP Biomedicals France Tel: Fax: Germany MP Biomedicals Phone: Fax: Japan MP Bio Japan K.K. Tel: Toll Free Tel: Fax: The Netherlands MP Biomedicals Netherlands Tel: Fax: Serbia MP Global d.o.o. Tel: Fax: Singapore MP Biomedicals Singapore Tel: Fax: Switzerland MP Biomedicals Switzerland Tel: Fax: United Kingdom MP Biomedicals UK Tel: Fax: Canada MP Biomedicals Canada Tel: Fax: Poland MP Biomedicals Poland Tel: Fax: MP Biomedicals Fountain Parkway Solon, OH tel: fax: