OneKit Genomic DNA Purification Handbook

Transcription

1 OneKit Genomic DNA Purification Handbook Genomic DNA Purification Kit (Blood/Cell) Genomic DNA Purificaiton Kit (Tissue) Genomic DNA Purification (Plant)

2 Notice to the User IMPORTANT! It is important that users read the entire manual before commencing work. OneKit Genomic DNA Extraction Kit 1

3 Warranty and Liability Phalanx Biotech Group s products are intended for research use only, and not intended for any other uses. OneKit products are designed and manufactured for research use only. Buyers and users agree and understand that they are not granted the right to use OneKit products for clinical diagnostic purposes unless they obtain written approval from the appropriate government authority. Phalanx Biotech Group (Phalanx Biotech) will not be liable for any damages arising from the use of its products in any manner other than their intended use or for the use of its products for clinical diagnostic purposes without written approval from the appropriate government authority. The manufacture, sale, or importation of products from Phalanx Biotech is not permitted without the prior written consent from Phalanx Biotech. Buyers and users agree and acknowledge that Phalanx Biotech is the owner and has the copyrights to the information of all OneKit products. Phalanx Biotech is founded on the mission to offer researchers high-quality and user-friendly solutions at an affordable price. Your satisfaction in using our products is very important to us. Therefore, if any of our products is not performing to the standard we promised, we are willing to replace the product, or credit the product purchase price. Phalanx Biotech accepts liability of ONLY the purchase price of its products, and has no other liabilities. 2 OneKit Genomic DNA Extraction Kit

4 Contact Information Asia Office Phalanx Biotech Group 6 Technology Road 5, 6 th Floor Hsinchu Science Park Hsinchu 30077, Taiwan Ph: FAX: Toll Free: (Taiwan) twservice@phalanxbiotech.com Web site: OneKit Genomic DNA Extraction Kit 3

5 Thank You Phalanx Biotech Group would like to extend special thanks to our customers who have provided feedback that enabled us to improve the Genomic DNA Purification Kit User Guide. 4 OneKit Genomic DNA Extraction Kit

6 Contents GETTING START... 6 KIT CONTENTS... 6 STORAGE CONDITION... 7 QUALITY CONTROL... 8 PRODUCT INTRODUCTION... 9 SPECIFICATION THE PURIFICATION PROCEDURE PROTOCOL Genomic DNA Purification Kit (Blood & Cell) Protocol Genomic DNA Purification Kit (Tissue) Protocol Genomic DNA Purification Kit (Plant) Protocol TROUBLESHOOTING REFERENCE ORDERING INFORMATION OneKit Genomic DNA Extraction Kit 5

7 Getting Start Phalanx Biotech Group Please read the introductory information below to help familiarize yourself with OneKit before use. Kit Contents Genomic DNA Purification Kit (Blood & Cell) (50) (100) Cat. No. GDB_0050 GDB_0100 OKD Columns RBC Lysis Buffer 100 ml 200 ml RDT Buffer 15 ml 30 ml LDT Buffer 15 ml 30 ml WD1 Buffer 25 ml 50 ml WD2 Buffer a (concentrate) 25 ml 25 ml ED Buffer 30 ml 30 ml Proteinase K b 11 mg 11 mg x 2 2 ml Collection Tubes Genomic DNA Purification Kit (Tissue) (50) (100) Cat. No. GDT_0050 GDT_0100 OKD Columns RDT Buffer 15 ml 30 ml LDT Buffer 15 ml 30 ml WD1 Buffer 25 ml 50 ml WD2 Buffer a (concentrate) 25 ml 25 ml ED Buffer 30 ml 30 ml Proteinase K b 11 mg 11 mg x 2 Micropestles ml Collection Tubes OneKit Genomic DNA Extraction Kit

8 Genomic DNA Purification Kit (Plant) (50) (100) Cat. No. GDP_0050 GDP_0100 OKD Columns LDP1 Buffer 25 ml 50 ml LDP2 Buffer 25 ml 50 ml LDP3 Buffer 6 ml 15 ml BDP Buffer c 15 ml 30 ml WD1 Buffer 25 ml 50 ml WD2 Buffer a (concentrate) 12.5 ml 25 ml ED Buffer 30 ml 30 ml RNase A (10mg/ml) 275 µl 550 µl Filter Column ml Collection Tubes a. Add 4 volumes of ethanol (96-100%) to1 volume of WD2 Buffer prior to initial use. b. Add 1.1 ml ddh 2 O to the tube and mix by vortexing. Store prepared Proteinase K (10 mg/ml) at 4 C. For long term storage, aliquot and store at -20 C. c. Add 2 volumes of Isopropanol to 1 volume of BDP Buffer prior to initial use. Buffers contain guanidine hydrochloride which is harmful and irritant. Wear a lab coat, disposable gloves and protective goggles when handling. Storage Condition OneKit Genomic DNA Purification Kit should be stored dry at room temperature (15-25 ). Under these conditions, the products can be stored for up to 12 months without showing any reduction in performance and quality. Note: RNase A should be stored at 4 C or - 20 C for long-term storage. Store prepared Proteinase K (10 mg/ml) at 4 C. For long term storage, aliquot and store at -20 C. OneKit Genomic DNA Extraction Kit 7

9 Quality Control Genomic DNA Purification Kit (Blood & Cell) The quality of Genomic DNA Purification Kit (Blood & Cell) is tested on a lot-to-lot basis. Kits are tested by isolation of genomic DNA from 200 µl human whole blood. The purified DNA was quantified with a spectrophotometer and the yield is 4-6 µg with A260/A280 ratio The quality of Genomic DNA Purification Kit (Tissue) The quality of Genomic DNA Purification Kit (Tissue) is tested on a lot-to-lot basis. Kits are tested by isolation of genomic DNA from 10 mg mouse liver. The purified DNA was quantified with a spectrophotometer and the yield is more than 10 µg with A260/A280 ratio Genomic DNA Purification Kit (Plant) The quality of the OneKit Genomic DNA Purification Kit (Plant) is tested on a lot-to-lot basis. The kits are tested by isolation of genomic DNA from 100mg young leaves. M Comparison OneKit and Other Brand 1. Arabidopsis thaliana (Other Brand) 2. Arabidopsis thaliana (OneKit) 3. Pisum sativum (Other Brand) 4. Pisum sativum (OneKit) 5. Populus tremula (Other Brand) 6. Populus tremula (OneKit) 7. Bambusa lodhamii Munro (Other Brand) 8. Bambusa lodhamii Munro (OneKit) 8 OneKit Genomic DNA Extraction Kit

10 Product Introduction Genomic DNA Purification Kit (Blood /Cells) OneKit Genomic DNA Purification Kit (Blood / Cells) provides a fast and economical method for purification of total DAN (including genomic, mitochondrial and viral DNA) from whole blood, plasma,serum, buffy coat, other body fluids, lymphocytes and cultures cells. The blood protocol utilities RBC Lysis Buffer, LDT Buffer and a rapid heating step to release DNA into solution. DNA in the chaotropic salt solution binds to the glass fiber matrix of column. After washing off the contaminants, the purified DNA is eluted by low salt elution buffer or water. There is no requirement for phenol/chloroform extraction or alcohol precipitation. Purified DNA of approximately kb is suitable for PCR or other enzymatic reactions. Genomic DNA Purification Kit (Tissue) OneKit Genomic DNA Purification Kit (Tissue) has been designed for purification of total DAN (including genomic, mitochondrial and viral DNA) from a variety of animal tissues or cells. The provided micropestle can efficiently homogenize tissue sample to shorten the time spent foe lysis. The method uses proteinase K and a chaotropic salt, guanidine hydrochloride to lysis cells and degrade protein. DNA in the chaotropic salt solution binds to the glass fiber matrix of column. After washing off the contaminants, the purified DNA is eluted by low salt elution buffer or water. Purified DNA of approximately kb in length is suitable for PCR or other enzymatic reactions. OneKit Genomic DNA Extraction Kit 9

11 Genomic DNA Purification Kit (Plant) OneKit Genomic DNA Purification Kit (Plant) provides a fast and simple method to isolate DNA from plant tissue and cells. In the first step, the sample is lysed by homogenization. The lysate is treated with RNase A to remove RNA. In the presence of achaotropic salt, the genomic DNA in the lysate binds to the glass matrix in the spin column. The contaminations are washed with an ethanol based wash buffer and purified genomic DNA is eluted by low salt elution buffer or water. The protocol does not require phenol extraction and alcohol precipitation. The entire procedure can be complete in 60 minutes. Two Buffer System Protocol: Plant species are extremely diverse in their metabolic components and might contain large amounts of lipids, polysaccharides, polyphenolics and proteins. Due to the reason, OneKit Genomic DNA Purification Kit (Plant) offers two lysis buffers for optimum performance. LDP1 Buffer: For most common plant species, uses LDP1 Buffer to lysis plant samples (standard protocol); this system ensures to purify DNA with high yields and little degradation. LDP2 Buffer: The detergent system with LDP2 Buffer is suitable to lysis plant samples which contain large amounts of polysaccharides. For most of plant species, both buffers give similar results. Researchers may try one buffer system first or both in parallel. 10 OneKit Genomic DNA Extraction Kit

12 Specification Genomic DNA Purification Kit (Blood & Cell) Protocol Genomic DNA Purification Kit (Blood & Cell) Sample Sources Whole blood, Cultured cell, Yeast, Gram negative/positive bacteria Sample Amount 300 µl Whole Blood, 10 7 Cultured Cells, 10 7 yeast, 10 9 Bacteria Cells Elution Volume µl Yield up to 50 µg Operation Time minutes Genomic DNA Purification Kit (Tissue) Protocol Genomic DNA Purification Kit (Tissue) Sample Sources Tissue, Paraffin-embedded tissue, Buccal Swab Sample Amount 20 mg Tissue, 0.5 cm Mouse Tail Elution Volume µl Yield up to 50 µg Operation Time minutes Genomic DNA Purification Kit (Plant) Protocol Genomic DNA Purification Kit (Plant) Sample Sources Plant tissue Sample Amount 100 mg plant tissue Elution Volume 50 µl Yield 20 µg Operation Time < 60 minutes Note: OneKit Genomic DNA Purification Kits are developed, designed and sold for research only. OneKit Genomic DNA Extraction Kit 11

13 The Purification Procedure 12 OneKit Genomic DNA Extraction Kit

14 Protocol Genomic DNA Purification Kit (Blood & Cell) Protocol For Fresh Blood Cell Notes: % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Blood RBC Lysis 1. Collect fresh blood in EDTA-Na 2 - treated collection tubes (or other anticoagulant mixture). 2. Apply up to 300 µl of blood to a 1.5 ml microcentrifuge If blood sample is more than 300 µl (up to 1 ml), apply the sample to a sterile 15 ml centrifuge 3. Add 3 volumes of RBC Lysis Buffer to 1 volume of the sample and mix by inversion. DO NOT vortex. 4. Incubate the tube for 5 minutes at room temperature. 5. Centrifuge for 2 minutes at 3,000 x g and discard the supernatant. 6. Add 200 µl RBC Lysis Buffer to resuspend the cell pellet. Cell Lysis 7. Add 200 µl LDT Buffer and 20 µl Proteinase K (10 mg/ml) to the tube and mix by vortexing. OneKit Genomic DNA Extraction Kit 13

15 8. Incubate at 60 C for 20 minutes until the sample lysate is clear. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 60 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 9. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting 10. Place an OKD Column into a 2 ml Collection Tube. 11. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 5 minutes. 12. Discard flow-through. Place the OKD Column back into the same Wash 13. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 14. Discard flow-through. Place the OKD Column back into the same 15. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 16. Discard flow-through. Place the OKD Column back into the same 17. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 14 OneKit Genomic DNA Extraction Kit

16 18. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 19. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 20. Centrifuge at 1,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 15

17 Genomic DNA Purification Kit (Blood & Cell) Protocol For Frozen Blood Notes: % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Cell Lysis 1. Add 20 µl Proteinase K (10 mg/ml) and 200 µl LDT Buffer to the 200 µl blood sample directly 2. Incubate at 60 C for 30 minutes until the sample lysate is clear. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 60 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 3. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting 4. Place an OKD Column into a 2 ml Collection Tube. 5. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 2 minutes. 6. Discard flow-through. Place the OKD Column back into the same 16 OneKit Genomic DNA Extraction Kit

18 Wash 7. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 8. Discard flow-through. Place the OKD Column back into the same 9. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 10. Discard flow-through. Place the OKD Column back into the same 11. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 12. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 13. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 14. Centrifuge at 6,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl OneKit Genomic DNA Extraction Kit 17

19 Genomic DNA Purification Kit (Blood & Cell) Protocol For Cultured Animal Cell Notes: % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Cell Harvesting 1. Transfer cells to a microcentrifuge tube (not provided) and harvest the cells with centrifugation for 20 seconds at 6,000 x g. When using adherent cells, trypsinize the cells before harvesting. 2. Discard the supernatant and resuspend the cells in 150 µl RBC Lysis Buffer. Cell Lysis 3. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample. 4. Incubate at 70 C for 10 minutes until the sample lysate is clear. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 70 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 5. Add 200 µl of ethanol (96-100%) to the sample lysate and 18 OneKit Genomic DNA Extraction Kit

20 vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting. 6. Place an OKD Column into a 2 ml Collection Tube. 7. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 2 minutes. 8. Discard flow-through. Place the OKD Column back into the same Wash 9. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 10. Discard flow-through. Place the OKD Column back into the same 11. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 12. Discard flow-through. Place the OKD Column back into the same 13. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 14. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 15. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 16. Centrifuge at 6,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 19

21 Genomic DNA Purification Kit (Blood & Cell) Protocol For Nucleated Erythrocyte Notes: % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Cell Harvesting 1. For nucleated erythrocytes (e.g. bird or fish), the sample volume can be up to 10 µl. 2. Add 150 µl RDT Buffer to a microcentrifuge tube and apply blood sample to the 3. Vortex to mix sample. Cell Lysis 4. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample. 5. Incubate at 70 C for 10 minutes until the sample lysate is clear. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 70 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 6. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate 20 OneKit Genomic DNA Extraction Kit

22 appears, break up by pipetting. 7. Place an OKD Column into a 2 ml Collection Tube. 8. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 2 minutes. 9. Discard flow-through. Place the OKD Column back into the same Wash 10. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 11. Discard flow-through. Place the OKD Column back into the same 12. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 13. Discard flow-through. Place the OKD Column back into the same 14. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 15. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 16. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 17. Centrifuge at 6,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 21

23 Genomic DNA Purification Kit (Blood & Cell) Protocol For Gram-Negative Bacteria Notes: % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Cell Harvesting 1. Transfer bacterial culture (<10 9 ) to a microcentrifuge tube (not provided). Centrifuge for 1 minute at 10,000 x g and discard the supernatant. 2. Add 200 µl of RDT Buffer to the tube and vortex or pipette to resuspend the cell pellet. Incubate at room temperature for 5 minutes. Cell Lysis 3. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample. 4. Incubate at 70 C for 10 minutes until the sample lysate is clear. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 70 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 5. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate 22 OneKit Genomic DNA Extraction Kit

24 appears, break up by pipetting. 6. Place an OKD Column into a 2 ml Collection Tube. 7. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 2 minutes. 8. Discard flow-through. Place the OKD Column back into the same Wash 9. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 10. Discard flow-through. Place the OKD Column back into the same 11. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 12. Discard flow-through. Place the OKD Column back into the same 13. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 14. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 15. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 16. Centrifuge at 6,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 23

25 Genomic DNA Purification Kit (Blood & Cell) Protocol For Gram-Positive Bacteria Notes: Prepare the lysozyme buffer fresh immediately prior to use. Lysozyme Buffer (20 mg/ml lysozyme, 20 mm Tris-HCl, 2 mm EDTA, 1% Triton X-100, ph8.0) % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Cell Harvesting 1. Transfer bacterial culture (<10 9 ) to a microcentrifuge tube (not provided). Centrifuge for 1 minute at 10,000 x g and discard the supernatant. 2. Add 200 µl of Lysozyme Buffer to the tube and vortex or pipette to resuspend the cell pellet. 3. Incubate at room temperature for 10 minutes. During incubation, invert the tube every 2-3 minutes. Cell Lysis 4. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample. Incubate at 70 C for 10 minutes until the sample lysate is clear. 5. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 70 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 24 OneKit Genomic DNA Extraction Kit

26 6. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting. 7. Place an OKD Column into a 2 ml Collection Tube. 8. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 2 minutes. 9. Discard flow-through. Place the OKD Column back into the same Wash 10. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 11. Discard flow-through. Place the OKD Column back into the same 12. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 13. Discard flow-through. Place the OKD Column back into the same 14. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 15. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 16. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 17. Centrifuge at 6,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 25

27 Genomic DNA Purification Kit (Blood & Cell) Protocol For Yeast Notes: Prepare Sorbitol Buffer (1.2 M sorbitol, 10 mm CaCI, 0.1 M Tris-Cl ph7.5, 35 mm β-mercaptoethanol) % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Cell Harvesting 1. Harvest yeast (up to 5 x 10 7 ) by centrifugation for 10 minutes at 5,000 x g. Discard the supernatant and resuspend the cell pellet in 600 µl sorbitol buffer. 2. Add 200U lyticase or zymolase. Incubate at 30 C for 30 minutes. Centrifuge the mixture for 10 minutes at 2,000 x g to harvest Spheroplast. 3. Remove the supernatant and add 200 µl of RDT Buffer to the tube and vortex or pipette to resuspend the cell pellet. Incubate at room temperature for 5 minutes. Cell Lysis 4. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample.incubate at 70 C for 10 minutes until the sample lysate is clear. 5. During incubation, invert the tube every 3 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 70 C dry bath (for DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. 26 OneKit Genomic DNA Extraction Kit

28 DNA Binding 6. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting. 7. Place an OKD Column into a 2 ml Collection Tube. 8. Apply the total mixture (including any precipitate) from previous step to the OKD column. Close the cap and centrifuge at 10,000 x g for 2 minutes. 9. Discard flow-through. Place the OKD Column back into the same Wash 10. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 11. Discard flow-through. Place the OKD Column back into the same 12. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 13. Discard flow-through. Place the OKD Column back into the same 14. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 15. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 16. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 17. Centrifuge at 6,000 x g for 30 seconds to elute purified DNA. Standard elution volume is 100 µl. If less sample is used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 27

29 Genomic DNA Purification Kit (Tissue) Protocol For Tissue Phalanx Biotech Group Notes: % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Sample Preparation 1. Cut up 20 mg of animal tissue (or 0.5 cm of mice tail) and transfer to a microcentrifuge tube (not provided). If tissue has a higher number of cells (e.g. spleen or liver), reduce starting material to 10 mg. 2. Use provided micropestle to grind the tissue to a pulp. 3. Add 200 µl RDT Buffer to the tube and continue to homogenize the sample tissue with grinding. Lysis 4. Add 20 µl Proteinase K (10 mg/ml) to the tube and mix by vortexing. 5. Incubate at 60 C for 30 minutes to lyse the sample. During incubation, invert the tube every 5 minutes. 6. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample. 7. Incubate at 70 C for 20 minutes until the sample lysate is clear. During incubation, invert the tube every 5 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 60 C dry bath (for DNA Elution). If there is insoluble material present following incubation, centrifuge at 10,000 x g for 2 minutes and transfer the supernatant to a new microcentrifuge tube (not provided). 28 OneKit Genomic DNA Extraction Kit

30 Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 8. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting. 9. Place an OKD Column into a 2 ml Collection Tube. 10. Apply the total mixture (including any precipitate) from the previous step to the OKD Column. Close the cap and centrifuge at 10,000 x g for 5 minutes. 11. Discard flow-through. Place the OKD Column back into the same Wash 12. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 13. Discard flow-through. Place the OKD Column back into the same 14. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 15. Discard flow-through. Place the OKD Column back into the same 16. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 17. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 18. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. OneKit Genomic DNA Extraction Kit 29

31 19. Centrifuge at 10,000 x g for 1 minute to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. 30 OneKit Genomic DNA Extraction Kit

32 Genomic DNA Purification Kit (Tissue) Protocol For Paraffin-Embedded Tissue Phalanx Biotech Group Notes: Xylene, % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Sample Preparation 1. Slice small section (up to 25 mg) from blocks of paraffin-embedded tissue and transfer to a microcentrifuge tube (not provided). 2. Add 1 ml xylene to each Vortex vigorously and incubate at room temperature for 10 minutes. Vortex occasionally during incubation. 3. Centrifuge at 10,000 x g for 3 minutes. Remove supernatant with a pipette. 4. Add 1 ml ethanol to wash sample pellet and mix by inverting. 5. Centrifuge at 10,000 x g for 3 minutes. Remove supernatant with a pipette. 6. Repeat the step 4 to Open tube and incubate at 37 C for 15 minutes to evaporate the residual ethanol. Lysis 8. Add 20 µl Proteinase K (10 mg/ml) to the tube and mix by vortexing. 9. Incubate at 60 C for 30 minutes to lyse the sample. During incubation, invert the tube every 5 minutes. 10. Add 200 µl LDT Buffer to the sample and vortex for 5 seconds to mix sample. OneKit Genomic DNA Extraction Kit 31

33 11. Incubate at 70 C for 20 minutes until the sample lysate is clear. During incubation, invert the tube every 5 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 60 C dry bath (for DNA Elution). If there is insoluble material present following incubation, centrifuge at 10,000 x g for 2 minutes and transfer the supernatant to a new microcentrifuge tube (not provided). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 12. Add 200 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting. 13. Place a OKD Column into a 2 ml Collection Tube. 14. Apply the total mixture (including any precipitate) from the previous step to the OKD Column. Close the cap and centrifuge at 10,000 x g for 5 minutes. 15. Discard flow-through. Place the OKD Column back into the same Wash 16. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 17. Discard flow-through. Place the OKD Column back into the same 18. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 19. Discard flow-through. Place the OKD Column back into the same 32 OneKit Genomic DNA Extraction Kit

34 20. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 21. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 22. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 23. Centrifuge at 10,000 x g for 1 minute to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 33

35 Genomic DNA Purification Kit (Tissue) Protocol For Buccal Swab Phalanx Biotech Group Notes: Swab, cotton, DACRON or C.E.P swabs, PBS, % ethanol and RNase A (10 mg/ml, for optional step) are required. Add ethanol to the buffer according to component instructions. Sample Preparation 1. Scrape the swab firmly against the inside of each cheek 6-7 times and air dry the swab (sample donor should not ingest anything at least 30 minutes prior to sample collection). 2. Separate the swab from the stick. Place the buccal swab into a 2 ml microcentrifuge tube and 500 µl PBS. Lysis 3. Add 20 µl Proteinase K (10 mg/ml) and 500 µl LDT Buffer to the sample and mix by vortexing. 4. Incubate at 70 C for 20 minutes to lyse the sample. During incubation, invert the tube every 5 minutes. At this time, preheat required ED Buffer (200 µl per sample) in a 60 C dry bath (for DNA Elution). If there is insoluble material present following incubation, centrifuge at 10,000 x g for 2 minutes and transfer the supernatant to a new microcentrifuge tube (not provided). DNA Binding 5. Add 500 µl of ethanol (96-100%) to the sample lysate and vortex immediately for 10 seconds to mix sample. If precipitate appears, break up by pipetting. 6. Place an OKD Column into a 2 ml Collection Tube. 7. Apply 700 µl mixtures (including any precipitate) from the 34 OneKit Genomic DNA Extraction Kit

36 previous step to the OKD column. 8. Close the cap and centrifuge at 10,000 x g for 1 minute. Discard flow-through. Place the OKD Column back into the same 9. Apply the remaining mixture to the OKD Column. Repeat Step 8. Wash 10. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 11. Discard flow-through. Place the OKD Column back into the same 12. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 13. Discard flow-through. Place the OKD Column back into the same 14. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. DNA Elution 15. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 16. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 17. Centrifuge at 10,000 x g for 1 minute to elute purified DNA. Standard elution volume is 100 µl. If fewer samples are used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. OneKit Genomic DNA Extraction Kit 35

37 Genomic DNA Purification Kit (Plant) Protocol For Plant Phalanx Biotech Group Notes: Add % ethanol to the buffer according to component instructions. LDP1 Buffer: For most common plant species, uses LDP1 Buffer to lysis plant samples (standard protocol); this system ensures to purify DNA with high yields and little degradation. LDP2 Buffer: The detergent system with LDP2 Buffer is suitable to lysis plant samples which contain large amounts of polysaccharides. Tissue Dissociation 1. Cut 50 mg (up to 100 mg) of fresh or frozen plant tissue or 5 mg (up to 100 mg) of dried sample. 2. Grind the sample with mortar and pestle under liquid nitrogen to a fine powder. For some plant samples, liquid nitrogen may be unnecessary for homogenization. 3. Transfer it into a microcentrifuge tube (not provided). Cell Lysis 4. Add 400 µl LDP1 Buffer (or LDP2 Buffer) and RNase A (10mg/ml) to the sample tube and mix by vortexing. DO NOT mix LDP1 (LDP2) Buffer with RNase A before use. 5. Incubate at 65 C for 10 minutes. Invert the tube every 5 minutes during incubation. At this time, preheat required ED Buffer (200 µl per sample) in a 65 C dry bath (for DNA Elution). 6. Add 100 µl LDP3 Buffer to the sample and mix by vortexing. 7. Incubate on ice for 3 minutes. Place a Filter Column into a 2 ml Collection Tube. 8. Apply the entire lysate from previous step to the Filter Column 36 OneKit Genomic DNA Extraction Kit

38 and centrifuge at 10,000 x g for 3 minutes. 9. Discard the Filter Column and carefully transfer clarified supernatant in the Collection Tube to a new 1.5 ml microcentrifuge tube (not provided). DNA Binding 10. Add 1.5 volume of BDP Buffer (isopropanol added) to the clear lysate and vortex immediately for 5 seconds to mix sample. For example, Add 750 µl BDP Buffer to 500 µl lysate. 11. Place an OKD Column into a 2 ml Collection Tube. 12. Apply 700 µl mixtures (including any precipitate) from previous step to the OKD column. Centrifuge at 10,000 x g for 2 minutes. 13. Discard flow-through. Place the OKD Column back into the same 14. Apply the remaining mixture to OKD Column. Centrifuge at 10,000 x g for 2 minutes and then discard flow-through. Place the OKD Column back into the same Wash 15. Add 400 µl of WD1 Buffer to the OKD Column. Centrifuge at 10,000 x g for 2 minutes. 16. Discard flow-through. Place the OKD Column back into the same 17. Add 600 µl of WD2 Buffer (ethanol added) to the OKD Column and centrifuge at 10,000 x g for 2 minutes. 18. Discard flow-through. Place the OKD Column back into the same 19. Centrifuge at 10,000 x g for 5 minutes to dry the column matrix. OneKit Genomic DNA Extraction Kit 37

39 Optional Step: Removing residue pigment If a few pigments remain on the column matrix, perform this procedure. a. After Wash Steps are completed, add 400 µl of ethanol in the OKD Column and centrifuge at 10,000 x g for 30 seconds. b. Discard flow-through. Place the OKD Column back into the same c. Centrifuge at 10,000 x g for 3 minutes to dry the column matrix. If RNA-free genomic DNA is required, perform this optional step. a. Add 5 µl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Elution 20. Transfer the dried OKD Column to a clean 1.5 ml microcentrifuge tube (not provided). 21. Add 100 µl of preheated ED Buffer to the center of the column matrix and let the column stand for 3-5 minutes until the liquid is absorbed by the matrix. 22. Centrifuge at 10,000 x g for 1 minute to elute purified DNA. Standard elution volume is 100 µl. If less sample volume is used, reduce the elution volume (30-50 µl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase recovery and the total elution volume to about 200 µl. 38 OneKit Genomic DNA Extraction Kit

40 Troubleshooting Phalanx Biotech Group For Genomic DNA Purification Kit (Blood / Cell ) 1. Column clogged A. Overloaded column with sample Reduce sample volume or separate into multiple tubes. B. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. 2. Low yield A. Incorrect DNA Elution Step Ensure that ED Buffer was added and absorbed to the centre of OKD Column matrix. B. Incomplete DNA Elution Elute twice to increase yield. 3. Eluted DNA does not perform well in downstream applications A. Residual ethanol contamination Following the wash step, dry OKD Column with additional centrifuge at full speed for 5 minutes or incubation at 60 for 5 minutes. B. RNA Contamination Perform optional RNA degradation step C. Protein Contamination Reduce the sample amount After DNA Binding Step, apply 400ul WD1 Buffer to wash OKD Column and centrifuge at 13,000 rpm at 13,000 for 30 seconds. Proceed with Wash Step. D. Genomic DNA was degraded OneKit Genomic DNA Extraction Kit 39

41 Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. For Genomic DNA Purification Kit (Tissue) 1. Column clogged A. Overloaded column with sample Reduce sample volume or separate into multiple tubes. B. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. 2. Low yield A. Incomplete DNA Elution Elute twice to increase yield. Incubate OKD Column for 5 min at room temperature before centrifuge during Elution Step. B. Incorrect or inappropriate WD2 Buffer or ED Buffer Ethanol must be added to WD2 Buffer before using and DNA will only be eluted efficiently in low-salt and weak-base preheated ED Buffer. C. Inefficient cell lysis due to incomplete mixing with LDT Buffer Be sure to mix sample and LDT Buffer immediately by vortexing. D. Insufficient proteinase activity or insufficient incubation time Be sure to store the proteinase solution at 4. Ensure the proteinase isn t added directly to LDT Buffer. After incubation, ensure that no residual particulates are visible. E. No ethanol or error percentage ethanol added to the lysate before loading into OKD column 40 OneKit Genomic DNA Extraction Kit

42 Repeat the procedure with a new sample. 3. Eluted DNA does not perform well in downstream applications A. Residual ethanol contamination Following the wash step, dry OKD Column with additional centrifuge at full speed for 5 minutes or incubation at 60 for 5 minutes. B. RNA Contamination Perform optional RNA degradation step. C. Protein Contamination Reduce the sample amount After DNA Binding Step, apply 400ul WD1 Buffer to wash OKD Column and centrifuge at 13,000 rpm at 13,000 for 30 seconds. Proceed with Wash Step. D. Genomic DNA was degraded Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. E. Elute DNA carrier salt residues Wash the column twice with WD2 Buffer. For Genomic DNA Purification Kit (Plant) 1. Column clogged A. Overloaded column with sample Reduce sample volume or separate into multiple tubes. B. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. 2. Low yield A. Incorrect DNA Elution Step OneKit Genomic DNA Extraction Kit 41

43 Ensure that ED Buffer was added and absorbed to the centre of OKD Column matrix. B. Incomplete DNA Elution Elute twice to increase yield 3. Eluted DNA does not perform well in downstream applications A. Residual ethanol contamination Following the wash step, dry OKD Column with additional centrifuge at full speed for 5 minutes or incubation at 60 for 5 minutes. B. RNA Contamination Perform optional RNA degradation step. C. Protein Contamination Reduce the sample amount. After DNA Binding Step, apply 400ul WD1 Buffer to wash OKD Column and centrifuge at 13,000 rpm for 30 seconds. Proceed with Wash Step. D. Genomic DNA was degraded Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. Reference 1. Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, OneKit Genomic DNA Extraction Kit

44 Ordering Information Phalanx Biotech Group Product Type Preps Product code Plasmid DNA Purification Kit mini 100 PDN_0100 mini 300 PDN_0300 midi 25 PDD_0025 maxi 10 PDX_0010 PCR Clean Up/Gel Extraction Kit 100 PGE_ PGE_0300 Genomic DNA Purification Kit Blood / Cultured 50 GDB_0050 Cell Blood / Cultured 100 GDB_0100 Cell Tissue 50 GDT_0050 Tissue 100 GDT_0100 Plant 50 GDP_0050 Plant 100 GDP_0100 Total RNA Purification Kit Blood / Cultured 50 TRB_0050 Cell Blood / Cultured 100 TRB_0100 Cell Tissue 50 TRT_0050 Tissue 100 TRT_0100 Plant 50 TRP_0050 Plant 100 TRP_0100 Viral Nucleic Acid Extraction Kit 50 VNE_0050 OneKit Genomic DNA Extraction Kit 43

45 OneKit User Guide 2009 Phalanx Biotech. All rights reserved.