For food testing purposes FOR IN VITRO USE ONLY Version 1, June 2015 For isolation of DNA from raw material and food products of plant and animal origin for PCR analysis Order No. S 400 06.1 Kit for 50 isolations Store the kit at 15 to 25 C 1
Table of Contents 1. What this Product Does... 3 1.1 Product Characteristics... 3 Number of Preparations... 3 Storage and Stability... 3 Kit Contents... 3 Chemical Hazard... 3 1.2 Additional Equipment and Reagents Required... 3 2. How to Use this Product... 4 2.1 Test Principle... 4 2.2 Basic Steps... 4 2.3 Application... 4 2.4 Sample Material... 4 2.5 Expected Yield... 4 2.6 Quality Control... 4 3. Procedures and Required Materials... 5 3.1 Before You Begin... 5 Preparation of Kit Working Solutions... 5 Sample Homogenization... 5 Caution... 5 3.2 Recommended Kits and Isolation Methods... 5 3.2 Isolation Procedures... 6 Procedure A: DNA Isolation for Analysis of Genetically Modified Organisms (GMO)... 6 Procedure B: DNA Isolation for Allergen Detection and Quantification Analysis... 7 Procedure C: DNA Isolation for Identification Analysis of Animal Species... 8 3.3 Storage of Samples... 9 4. Typical Results... 9 Purity and Integrity... 9 5. Appendix... 9 5.1 Troubleshooting... 9 5.2 Helpful Hints... 10 Centrifugation... 10 5.3 Reference... 10 6. Supplementary Information...10 6.1 Ordering Information... 10 6.2 Trademarks... 10 6.3 Contact and Support... 10 7. Change Index...10 2
1. What this Product Does 1.1 Product Characteristics Number of Preparations 50 isolations. Storage and Stability The components must be stored at 15 to 25 C. Kit components are guaranteed to be stable until the expiration date printed on the label. Note: Improper storage at 2 to 8 C (refrigerator) or -15 to -25 C (freezer) will adversely impact DNA purification when precipitates form in the solutions. After dissolution of Proteinase K the solution should be aliquoted and stored at -15 to -25 C. The solution is stable at -15 to -25 C for 12 months. Kit Contents All solutions are clear, and should not be used when precipitates have formed. If precipitates have formed, simply warm the solutions at 15 to 25 C or in a 37 C water bath until the precipitates have dissolved. Vial Cap Color Label Contents / Function 1 red 2 green 3 blue 4 colorless 5 purple Extraction Buffer Binding Buffer Wash Buffer Elution Buffer Proteinase K 2 x 45 ml For extraction of DNA 25 ml For binding of DNA to glass fiber fleece 10 ml, add 40 ml absolute ethanol For removing impurities 44 ml For elution of DNA Lyophilizate 100 mg For protein digestion and inactivation of endogenous nucleases 6 Filter Tubes Bag with 50 polypropylene tubes with two layers of glass fiber fleece, for use of up to 700 µl sample volume 7 Collection Tubes 3 x one bag with 50 polypropylene tubes (2 ml) Chemical Hazard The Extraction Buffer (vial 1) and the Binding Buffer (vial 2) contain irritating compounds that are harmful when brought in contact with skin, inhaled, or swallowed. Always store and use the buffers away from food for humans and animals. Always wear gloves, and follow standard safety precautions during handling. 1.2 Additional Equipment and Reagents Required Ethanol, absolute Isopropanol, absolute Water, double-distilled Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force (e.g., Eppendorf 5415C or equivalent) Microcentrifuge tubes, 1.5 ml and 2.0 ml, sterile Heating Unit Materials to disrupt and homogenize samples (e.g., mortar and pestle or bead mills) 3
2. How to Use this Product 2.1 Test Principle Following sample homogenization, the DNA is extracted at high temperature with the provided extraction buffer. After clearing the lysis mixture by centrifugation and Proteinase K digestion, the DNA binds selectively to special glass fibers pre-packed in the filter tube (1). Bound DNA is purified in two wash-and-spin steps to remove potential PCR inhibitors. Finally, a low salt elution releases the DNA from the glass fiber. This simple method eliminates the need for organic solvent extractions and DNA precipitation, thus providing rapid, simultaneous purification of many samples. 2.2 Basic Steps Stage Description 1 DNA is extracted by incubation with the Extraction Buffer. 2 Proteinase K digestion of endogenous nucleases and other impurities. 3 DNA is bound to the glass fibers pre-packed in the filter tube. 4 Washing of bound DNA, and purification from salts, proteins, and other cellular impurities. 5 Purified DNA is recovered using the Elution Buffer. 2.3 Application The is optimized for the isolation of DNA from various food samples (raw material and processed food) of plant and animal origin. The quality of the DNA obtained with the kit is highly suitable for qualitative and quantitative applications using any PCR system. Note: The recommended amplicon length for PCR analysis is < 200 bp because the DNA of processed food may be degraded to a certain extent. 2.4 Sample Material 200 mg of homogenized food sample (raw material or processed food). Note: Additional aliquots can be prepared and the DNA concentrated by loading them step-by-step into one Filter Tube. This procedure is recommended if the DNA is degraded due to extensive processing of the food sample during production (e.g., heat, pressure), or if the DNA content of the food sample is low. 2.5 Expected Yield Typically, 0.1 to 10 µg nucleic acids per 200 mg sample. Note: The obtained yield strongly depends on the processed food type. 2.6 Quality Control DNA of Roundup Ready soybean flour is extracted and purified as described below. 5 µl eluate is analyzed using the foodproof GMO Screening Kit (Order No. R 300 17). As expected, the resulting amplification signal is obtained at a cp-value < 25. The absence of contaminating DNA is controlled by an additional DNA preparation and a subsequent PCR test using the foodproof Plant Gene Detection Mix without adding soybean flour as the sample material. As expected, no amplification product is obtained. 4
3. Procedures and Required Materials 3.1 Before You Begin Preparation of Kit Working Solutions In addition to the ready-to-use solutions supplied with the kit, you will need the following working solutions; preparation of working solutions is required: Bottle / Cap Color Content Preparation of Working Solution Storage and Stability 3 blue foodproof Sample Preparation Kit III Wash Buffer Add 40 ml absolute ethanol to Wash Buffer. Note: Label and date bottle after ethanol is added. Store at 15 to 25 C Stable until the expiration date printed on kit label. 5 purple foodproof Sample Preparation Kit III Proteinase K Dissolve Proteinase K in 5 ml double-distilled water, aliquot solution. Store at 15 to 25 C, stable for 12 months. Sample Homogenization Heterogeneous food samples (e.g., not flour or liquid samples) must be homogenized in order to obtain a representative sample for analyzing and enhancing the efficiency of DNA extraction. Suitable methods for homogenization are grinding with a mortar and pestle or usage of commercial products, such as a mixer, homogenizer, or bead mills. The most suitable method depends on the food type analyzed. Caution Use sterile disposable polypropylene tubes and filter tips in order to avoid cross-contamination. Always wear gloves during the assay, and follow safety precautions to minimize contact when handling. 3.2 Recommended Kits and Isolation Methods Analytic Method Recommended Kits Recommended DNA Isolation Method GMO Screening GMO Identification GMO Quantification foodproof GMO Screening, Identification and Quantification Kits and LyoKits Procedure A: For Analysis of Genetically Modified Organisms (GMO) Allergen Detection Allergen Quantification foodproof Allergen Detection Kits Procedure B: For Allergen Detection and Quantification Analysis Animal Identification foodproof Animal Detection Kits and LyoKits Procedure C: For Identification Analysis of Animal Species 5
3.2 Isolation Procedures Procedure A: DNA Isolation for Analysis of Genetically Modified Organisms (GMO). The following protocol describes the isolation of DNA from 200 mg homogenized sample. The isolated DNA can be used for GMO screening, identification and quantification purposes. Step Action Volume Time / g Time / Temp. 1 Add Extraction Buffer (bottle 1, red cap) to 200 mg homogenized sample (in 2 ml microcentrifuge tubes). Vortex for 30 s. Note: Mix 2-3 times during the incubation by inverting the tube. If the matrix absorbs the Extraction Buffer, add additional buffer. 1 ml 30 s 80 C for 30 min 2 Centrifuge. 10 min at 12,000 x g 3 Add Binding Buffer (bottle 2, green cap) to a new 2 ml microcentrifuge tube. 400 µl 4 Transfer supernatant to the new 2 ml microcentrifuge tube with Binding Buffer, then mix gently but thoroughly by pipetting up and down. Add Proteinase K working solution (100 mg/5 ml ddh 2 O) Mix gently but thoroughly by pipetting up and down. 5 Add Isopropanol. Mix well by pipetting up and down. 6 Pipet the mixture into the upper reservoir of a combined Filter Tube-Collection Tube assembly. 600 µl 80 µl 200 µl 650 µl 72 C for 10 min 7 Centrifuge in a microcentrifuge. 8 Discard flow-through and Collection Tube, place Filter Tube in new Apply the remaining mixture to the same Filter Tube and centrifuge. Note: If DNA must be pooled, steps 6-7 can be repeated with additional sample preparations. 9 Discard flow-through and Collection Tube, place Filter Tube in new 10 Discard the flow-through and reuse the 11 Discard the flow-through and reuse the Centrifuge to remove residual Wash Buffer. 12 Insert Filter Tube in a clean 1.5 ml reaction tube. Add pre-warmed (70 C) Elution Buffer (bottle 4, colorless cap) onto the glass fibre fleece. 200 µl 10 s at max speed (13,000 x g) 15-25 C for 5 min 13 Centrifuge. 1 min at 5,000 g 14 The microcentrifuge tube now contains the eluted DNA. 6
Procedure B: DNA Isolation for Allergen Detection and Quantification Analysis The following protocol describes the isolation of DNA from 200 mg homogenized sample. The isolated DNA can be used for allergen detection and quantification purposes. Step Action Volume Time/g Time/Temp. 1 Add Extraction Buffer (bottle 1, red cap) to 200 mg homogenized sample (in 2 ml microcentrifuge tubes). Vortex for 30 s. Note: Mix 2-3 times during the incubation by inverting the tube. If the matrix absorbs the Extraction Buffer, add additional buffer. 1.5 ml 30 s 80 C for 30 min 2 Centrifuge. 10 min at 12,000 x g 3 Add Binding Buffer (bottle 2, green cap) to a new 2 ml microcentrifuge tube. 400 µl 4 Transfer supernatant to the new 2 ml microcentrifuge tube with Binding Buffer, and then mix gently but thoroughly by pipetting up and down. Add Proteinase K working solution (100 mg/5 ml ddh 2O) Mix gently but thoroughly by pipetting up and down. 5 Add Isopropanol. Mix well by pipetting up and down. 6 Pipet the mixture into the upper reservoir of a combined Filter Tube-Collection Tube assembly. 600 µl 80 µl 200 µl 650 µl 72 C for 10 min 7 Centrifuge in a microcentrifuge. 8 Discard flow-through and Collection Tube, place Filter Tube in new Apply the remaining mixture to the same Filter Tube and centrifuge. Note: If DNA must be pooled, steps 6-7 can be repeated with additional sample preparations. 9 Discard flow-through and Collection Tube, place Filter Tube in new 10 Discard the flow-through and reuse the 11 Discard the flow-through and reuse the Centrifuge to remove residual Wash Buffer. 12 Insert Filter Tube in a clean 1.5 ml reaction tube. Add pre-warmed (70 C) Elution Buffer (bottle 4, colorless cap) onto the glass fibre fleece. 100 µl 10 s at max speed (13,000 x g) 15-25 C for 5 min 13 Centrifuge. 1 min at 5,000 g 14 The microcentrifuge tube now contains the eluted DNA. 7
Procedure C: DNA Isolation for Identification Analysis of Animal Species The following protocol describes the isolation of DNA from 200 mg homogenized sample. The isolated DNA can be used for animal identification purposes. Step Action Volume Time/g Time/Temp. 1 Add Extraction Buffer (bottle 1, red cap) to 200 mg homogenized sample (in 2 ml microcentrifuge tubes). Vortex for 30 s. Add Proteinase K working solution (100 mg/5 ml ddh 2O). Note: Mix 2-3 times during the incubation by inverting the tube. If the matrix absorbs the Extraction Buffer, add additional buffer. 1 ml 80 µl 30 s 72 C for 30 min 2 Centrifuge. 10 min at 12,000 x g 3 Add Binding Buffer (bottle 2, green cap) to a new 2 ml microcentrifuge tube. Add Isopropanol. 4 Transfer supernatant to the new 2 ml microcentrifuge tube with Binding Buffer and Isopropanol, then mix gently but thoroughly by pipetting up and down. 5 Pipet the mixture into the upper reservoir of a combined Filter Tube- Collection Tube assembly. 400 µl 200 µl 600 µl 650 µl 6 Centrifuge in a microcentrifuge. 7 Discard flow-through and Collection Tube, place Filter Tube in new Apply the remaining mixture to the same Filter Tube and centrifuge Note: If DNA must be pooled, steps 6-7 can be repeated with additional sample preparations. 8 Discard flow-through and Collection Tube, place Filter Tube in new 9 Discard the flow-through and reuse the 10 Discard the flow-through and reuse the Centrifuge to remove residual Wash Buffer. 11 Insert Filter Tube in a clean 1.5 ml reaction tube. Add pre-warmed (70 C) Elution Buffer (bottle 4, colorless cap) onto the glass fiber fleece. 200 µl 10 s at max speed (13,000 x g) 15-25 C for 5 min 12 Centrifuge. 1 min at 5,000 g 13 The microcentrifuge tube now contains the eluted DNA. 8
3.3 Storage of Samples IF you want to Continue Stop THEN Use the eluated DNA directly. Store the DNA at 2 to 8 C or for later analysis at 15 to 25 C 4. Typical Results Purity and Integrity Purified DNA is free of other cellular components and DNA polymerase inhibitors. Note: The length of DNA fragments depends on the food type analyzed. DNA of highly processed food samples may be <1000 bp in length. 5. Appendix 5.1 Troubleshooting Problem Possible Cause Recommendation Low DNA yield or purity Absorbency (A 260) reading of product too high Sample pops out of wells in agarose gels Kit stored under nonoptimal conditions. Buffers or other reagents were exposed to conditions that reduce their effectiveness. Ethanol not added to Wash Buffer. Reagents and samples not completely mixed. Homogenization of food sample not sufficient. Glass fibers that can coelute with DNA scatter light. Eluate containing the purified DNA product is contaminated with ethanol from the Wash Buffer. Store the kit at 15-25 C at all times upon arrival. Store all buffers at 15-25 C. Close all reagent bottles tightly after each use to preserve ph, stability, and freedom of contamination. After any lyophilized reagent is reconstituted, aliquot it, then store the aliquot at 15 to 25 C. Add absolute ethanol to the Wash Buffer before using. After adding ethanol, mix the Wash Buffer well, and store at 15 to 25 C. Always mark Wash Buffer bottle to indicate the addition of ethanol. Always mix the sample tube well after addition of each reagent. Use a mortar and pestle or a commercial product, such like mixer or bead mills for disruption/homogenization. After elution step is complete, remove filter from tube containing eluted sample and spin sample tube for 2 min at maximum speed. Transfer supernatant into a new tube without disturbing the glass fibers at the bottom of the original tube. After the last wash step, make certain the flow-through containing Wash Buffer does not contact the bottom of the Filter Tube. If this has occurred, empty collection tube, re-insert the contaminated filter tube, and re-centrifuge for 30 s. 9
S 400 06.1 20 (1) 5.2 Helpful Hints Centrifugation For convenience, the following table shows corresponding centrifugal forces (g) for selected rotations per minute (rpm) when working with a standard table top microcentrifuge (e.g., such as Eppendorf 5415 C). Rotations per minute [rpm] Centrifugation force [g] 14,000 15,800 12,000 11,600 10,000 8,000 8,000 5,200 5,000 2,000 3,000 720 1,000 80 5.3 Reference 1 Vogelstein, B. & Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615 619 6. Supplementary Information 6.1 Ordering Information BIOTECON Diagnostics is offering a broad range of reagents and services. For a complete overview and for more information, please visit our website at www.bc-diagnostics.com. 6.2 Trademarks foodproof is a trademark of BIOTECON Diagnostics GmbH. Roundup Ready is a trademark of the Monsanto Company, St. Louis, MO, USA. Other brand or product names are trademarks of their respective holders. 6.3 Contact and Support If you have questions or experience problems with this or any other product of BIOTECON Diagnostics, please contact our Technical Support staff (for details see www.bc-diagnostics.com). Our scientists commit themselves to providing rapid and effective help. We also want you to contact us if you have suggestions for enhancing our product performance or using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to us and the world-wide research community. 7. Change Index Version 1, June 2015: First version of the package insert BIOTECON Diagnostics GmbH Hermannswerder 17 14473 Potsdam Germany Phone +49 (0) 331 2300-200 Fax +49 (0) 331 2300-299 www.bc-diagnostics.com bcd@bc-diagnostics.com 10