Genomic DNA Extraction kit Protocol Book

Transcription

1 Quality Nucleic Acid Purification System Genomic DNA Extraction kit Protocol Book Blood/Bacteria/Cultured Cells YGB50 // YGB100 // YGB300 // YGBM10 // YGBM25 Tissue YGT50 // YGT100 Plant YGP50 // YGP100 // YGPM10 // YGPM25 Ver

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3 Precautions I) Handling Requirements Do not use a kit after its expiration date has passed. Some reagents contain the hazardous compounds guanidine thiocyanate or guanidine hydrochloride. Do not let these reagents touch your skin, eyes, or mucous membranes. If contact does occur, wash the affected area immediately with large amounts of water. If you spill the reagents, dilute the spill with water before wiping it up. Do not allow reagents containing guanidine thiocyanate to mix with sodium hypochlorite solution or strong acids. This mixture can produce a highly toxic gas. II) Laboratory Procedures Handle all samples and the resulting waste as if potentially infectious, using safe laboratory procedures. As the sensitivity and titer of potential pathogens in the sample material varies, the operator has to optimize pathogen inactivation by the Lysis Buffer or take appropriate measures according to local safety regulations. RBC Bioscience does not warrant that samples treated with Lysis Buffer are completely inactivated and non-infectious. After sample processing is completed, remove and autoclave all disposable plastics, if you worked with potentially infectious sample material. Do not eat, drink or smoke in the laboratory work area. Do not pipette by mouth. Wear protective disposable gloves, laboratory coats and eye protection when handling samples and kit reagents. Do not use sharp or pointed objects when working with the reagent cartridge, in order to prevent damage of the sealing foil and loss of reagent. Do not contaminate the reagents with bacteria, virus, or ribonuclease. Use disposable pipettes and RNase-free pipette tips only to remove aliquots from reagent bottles. Use the general precautions described in the literature. Wash hands thoroughly after handling samples and test reagents. III) Waste Handling Discard unused reagents and waste in accordance with country, federal,state and local regulations.

4 CONTENTS Genomic DNA Extraction Kit Mini Blood/Bacteria/Cultured Cells 1 Cat.No. YGB50//YGB100//YGB300 Blood Protocol Cultured Cells Protocol Bacterial Protocol Yeast Protocol Troubleshooting Genomic DNA Extraction Kit Maxi Blood/Bacteria/Cultured Cells Cat.No. YGBM10//YGBM25 Fresh Blood Protocol Frozen Blood Protocol Troubleshooting

5 Genomic DNA Extraction Kit Mini Tissue 21 Cat.No. YGT50//YGT100 Tissue Protocol Troubleshooting Genomic DNA Extraction Kit Mini Plant Cat.No. YGP50//YGP100 Plant Protocol Troubleshooting Genomic DNA Extraction Kit Maxi Plant Cat.No. YGPM10//YGPM25 Plant Protocol 41

6 Genomic DNA Extraction Kit Mini Blood/Bacteria/Cultured Cells Cat.No. YGB50//YGB100//YGB300 Kit Contents Cat.No. YGB50 50 mini preps / kit RBC Lysis Buffer...100ml GB Buffer...15ml GT Buffer...15ml W1 Buffer... 25ml Wash Buffer (Concentrated)*...25ml Elution Buffer...30ml Proteinase K **...11 mg GD Column...50 pcs 2 ml Collection Tube...50 pcs Cat.No. YGB mini preps / kit RBC Lysis Buffer...200ml GB Buffer...30ml GT Buffer...30ml W1 Buffer...50ml Wash Buffer (Concentrated) *...25ml Elution Buffer...30ml Proteinase K **...11 mg x 2 GD Column pcs 2 ml Collection Tube pcs Cat.No. YGB mini preps / kit RBC Lysis Buffer...500ml GB Buffer...100ml GT Buffer...100ml W1 Buffer...130ml Wash Buffer (Concentrated)*... 40ml Elution Buffer...60ml Proteinase K **...11 mg x 6 GD Column pcs 2 ml Collection Tube pcs Sample (Protocols Included): 300 μl Whole Blood (up to 1ml),10 7 Cultured Cells,10 9 Bacteria (Gram +/-),10 7 Yeast. Average Yields: 300μl Whole Blood - 6μg, 200 μl Buffy - 50μg, 5 x 10 6 Lymph./Cult. Cells - 50μg. * Add 4 times volume of ethanol(96-100%) to Wash Buffer prior to initial use. ** Add 1.1 ml ddh 2 O to the tube and mix by vortexing. Store prepared Proteinase K (10mg/ml) at 4 C. For long term storage, aliquot and store at -20 C. Additional Requirement: Microcentrifuge Tube, Ethanol (96-100%), RNase A (10 mg/ml). 1 Genomic DNA Extraction Kit - Mini - Blood/Bacteria/Cultured Cells

7 Description Genomic DNA Extraction Mini Kit (Blood/Bacteria/Cultured Cells) provides a fast and economical method for purification of total DNA (including genomic, mitochondrial and viral DNA) from whole blood, plasma, serum, buffy coat, other body fluids, lymphocytes and cultured cells. This kit is also ideal for bacterial and cultured cells. The blood protocol utilises RBC lysis buffer, GB 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. Quality Control The quality of Genomic DNA Mini Kit (Blood/Bacteria/Cultured Cells) is tested on a lot-to-lot basis. The kits are tested by isolation of genomic DNA from 200μl of human whole blood. The purified DNA is quantified by spectrophotometer and the yield of genomic DNA is 4-6μg with A /A ratio Reference Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615. Note * For research use only. Not for use in diagnostic or therapeutic procedures. * Buffer contains guanidine hydrochloride which is harmful and can act as an irritant. During operation, always wear a lab coat, disposable gloves and protective goggles. Genomic DNA Extraction Kit - Mini - Blood/Bacteria/Cultured Cells 2

8 60 C 20min RBC Lysis Buffer GB Buffer / Proteinase K DNA Binding Wash Step Elution Step Purified DNA 3 Genomic DNA Extraction Kit - Mini - Blood Protocol

9 Blood Protocol RBC Lysis Buffer is provided to remove non-nucleated red blood cells and reduce hemoglobin contamination. But when the blood sample is less than 50μl or sample consists of nucleated blood cells, the Cells Protocol is recommended to purify DNA. Fresh Blood RBC Lysis 1. Collect fresh blood in EDTA-Na 2 treated collection tubes (or other anticoagulant mixture). 2. Apply up to 300μl of blood to a 1.5ml microcentrifuge tube. If blood sample is more than 300μl (up to 1 ml), apply the sample to a sterile 15ml centrifuge tube. 3. Add 3 times the sample volume of RBC Lysis Buffer 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 200μl RBC Lysis Buffer to resuspend the cell pellet. Cell Lysis 7. Add 200μl GB Buffer and 20μl Proteinase K (10mg/ml) to the tube and mix by vortexing. 8. Incubate at 60 C for 20 minutes until the sample lysate is clear. During incubation, invert the tube every 3 min. At this time, preheat required Elution Buffer (200μl per sample) in a 60 C water bath (For DNA Elution Step). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a.add 5μl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b.incubate at room temperature for 5 minutes. Genomic DNA Extraction Kit - Mini - Blood Protocol 4

10 DNA Binding 9. Add 200μl 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 a GD Column on a 2ml Collection Tube. 11. Apply the total mixture (including any precipitate) from previous step to the GD Column. Close the cap and centrifuge at 10,000 x g (13,000 rpm) for 5 minutes. 12. Discard the flow-through and return the GD column to the 2ml Collection Tube. Wash 13. Add 400μl of W1 Buffer to the GD Column. Centrifuge at10,000 x g (13,000 rpm) for 30 seconds. 14. Discard the flow-through and return the GD Column to the 2ml Collection Tube. 15. Add 600μl of Wash Buffer (ethanol added) in the GD Column. 16. Centrifuge at 10,000 x g (13,000 rpm) for 30 seconds. 17. Discard the flow-through and return the GD Columnto the 2ml Collection Tube. 18. Centrifuge at 10,000 x g (13,000 rpm) for 3 minutes to dry the column matrix. DNA Elution Standard elution volume is 100μl. If less sample volume is used, reduce the elution volume (30-50μl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution Step to increase DNA recovery and the total elution volume to about 200μl. 19. Transfer dried GD Column into a clean 1.5 ml microcentrifuge tube (not provided). 20. Add 100μl of preheated Elution Buffer to the centre of the column matrix. 21. Allow to stand for 3-5 minutes until Elution Buffer is absorbed by the matrix. 22. Centrifuge at 10,000 x g (13,000 rpm) for 30 seconds to elute purified DNA. 5 Genomic DNA Extraction Kit - Mini - Blood Protocol

11 Frozen Blood Protocol μl Blood + 20μl Proteinase K (10mg/ml) + 200μl GB Buffer, Incubate at 60 C for 30 min. 2. At this time, preheat required Elution Buffer (200μl per sample) in a 60 C water bath (For DNA Elution Step). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a.add 5μl of RNase A (10 mg/ml) (not provided) to sample lysate and vortex to mix. b.incubate at room temperature for 5 minutes. 3. Proceed with Fresh Blood Protocol Step 9 - DNA Binding. Genomic DNA Extraction Kit - Mini - Blood Protocol 6

12 Cultured Cells Protocol Additional Requirement: Microcentrifuge tube, Ethanol (96-100%), RNase A (10mg/ml) Sample Preparation Cultured Animal Cells: If using adherent cells, trypsinize the cells before harvesting. 1. Transfer cells to a microcentrifuge tube (not provided) and harvest the cells with centrifugation at 2,000 rpm for 5 min. 2. Discard the supernatant and resuspend the cells in 150μl RBC Lysis Buffer. Nucleated Erythrocytes: For nucleated erythrocytes (e.g. bird or fish), the sample volume can be up to 10μl. 1. Add 150μl GT Buffer to a microcentrifuge tube and apply blood sample to tube. 2. Vortex to mix sample. Lysis 3. Add 200μl GB Buffer to the sample. 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 Elution Buffer (200μl per sample) at 70 C.DNA Binding Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform the optional step. a. After 70 C incubation, add 5μl of RNase A (10mg/ml, provided by user) to sample lysate and vortex to mix sample. b. Incubate at room temperature for 5 minutes. 7 Genomic DNA Extraction Kit - Mini - Cultured cells Protocol

13 DNA Binding 5. Add 200μl 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 a GD Column on a 2 ml Collection Tube. 7. Apply all the mixture (including any precipitate) from previous step to the GD Column. 8. Close the cap and centrifuge at 10,000 x g (13,000 rpm) for 2 minutes. 9. Discard the flow-through and return the GD column to the 2ml collection tube Wash 10. Add 400μl of W1 Buffer to the GD Column. Centrifuge at 10,000 x g (13,000 rpm) for 30 seconds. 11. Discard the flow-through and return the GD Column to the 2ml Collection Tube. 12. Add 600μl of Wash Buffer (ethanol added) to the GD Column. Centrifuge at10,000 x g (13,000 rpm) for 30 seconds. 13. Discard the flow-through and return the GD Column to the 2ml Collection Tube 14. Centrifuge at full speed for 3 minutes to dry the column matrix. DNA Elution Standard elution volume is 100μl. If less sample to be used, reduce the elution volume (30-50μl) to increase DNA concentration. If higher DNA yield required, repeat the DNA Elution Step to increase DNA recovery and the total elution volume to about 200μl. 15. Transfer dried GD Column into a clean 1.5ml microcentrifuge tube (not provided). 16. Add 100μl of preheated Elution Buffer to the centre of the column matrix. 17. Allow to stand for 2 minutes until Elution Buffer is absorbed by the matrix. 18. Centrifuge at 10,000 x g (13,000 rpm) for 30 seconds to elute purified DNA. Genomic DNA Extraction Kit - Mini - Cultured cells Protocol 8

14 Bacterial Protocol Additional Requirements : Lysozyme Buffer, Microcentrifuge tube, RNase A (10mg/ml) Gram Negative 1. Transfer bacterial culture (<10 9 ) to a microcentrifuge tube (not provided). Centrifuge for 1 min at 10,000 x g (13,000 rpm) and discard the supernatant. 2. Add 200μl of GT Buffer to the tube and vortex or pipette to resuspend the cell pellet. Incubate at room temperature for 5 minutes. 3. Proceed to Cultured Cells Protocol Step 3 Lysis. Gram Positive Prepare Lysozyme Buffer: (20mg/ml lysozyme; 20mM Tris-HCl; 2mM EDTA; 1% Triton X-100; ph 8.0), prepare the Lysozyme Buffer fresh immediately prior to use. 1. Transfer bacterial culture (<10 9 ) to a microcentrifuge tube (not provided). Centrifuge for 1 min at 10,000 x g (13,000 rpm) and discard the supernatant. 2. Add 200μl of Lysozyme Buffer to the tube and vortex or pipette to resuspend the cell pellet. Incubate at room temperature for 10 minutes. During incubation, invert tube every 2-3 minutes. 3. Proceed to Cultured Cells Protocol Step 3 Lysis. 9 Genomic DNA Extraction Kit - Mini - Bacteria Protocol

15 Yeast Protocol Additional requirements: Sorbitol Buffer, Lyticase or Zymolase, Microcentrifuge tube, Ethanol (96-100%), RNase A (10mg/ml) Prepare Sorbitol Buffer: 1.2 M sorbitol; 10mM CaCl 2 ; 0.1M Tris-Cl ph 7.5, 35mM Beta-mercaptoethanol 1. Harvest yeast cells (up to 5x10 7 ) by centrifugation for 10 minutes at 5000 x g. Discard the supernatant and resuspend the cell pellet in 600μl sorbitol buffer. 2. Add 200U Lyticase or Zymolase. Incubate at 30 C for 30 minutes. Centrifuge the mixture for 10 min at 2,000 x g to harvest Spheroplast. 3. Remove the supernatant and add 200μl of GT Buffer to the tube and vortex or pipette to resuspend the cell pellet. Incubate at room temperature for 5 minutes. 4. Proceed to Cultured Cells Protocol Step 3 Lysis. Genomic DNA Extraction Kit - Mini - Yeast Protocol 10

16 Troubleshooting Problem Column clogged Possible Reason/Solution Overloaded column with sample Reduce sample volume or separate into multiple tubes. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. Low yield Incorrect DNA Elution Step Ensure that Elution Buffer was added and absorbed to the centre of GD Column matrix. Incomplete DNA Elution Elute twice to increase yield Eluted DNA does not perform well in downstream applications Residual ethanol contamination Following the wash step, dry GD Column with additional centrifugation at full speed for 5 minutes or incubation at 60ºC for 5 minutes. RNA Contamination Perform optional RNA degradation step. Protein Contamination Reduce the sample amount After DNA Binding Step, apply 400 μl W1 Buffer to wash GD Column and centrifuge at 13,000 rpm for 30 seconds. Proceed with Wash Step Genomic DNA was degraded Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. 11 Genomic DNA Extraction Kit - Mini - Blood/Bacteria/Cultured Cells

17 Brand Q RBC Real Genomics RBC Bioscience Labs Comparison: Genomic DNA Mini Kit (Blood/Bacteria/Cultured Cells) vs Brand Q Genomic DNA Extraction Kit Sample: 2 x 10 9 gram negative bacteria Lane 2-3 Brand Q DNA Mini Kit Lane 4-5 RBC Real Genomics RBC Bioscience Labs Genomic DNA Extraction Kit - Mini - Blood/Bacteria/Cultured Cells 12

18 Genomic DNA Extraction Kit Maxi Blood/Bacteria/Cultured Cells Cat.No. YGBM10//YGBM25 Kit Contents Cat.No. YGBM10 10 Maxi preps / kit RBC Lysis Buffer...200ml x2 GB Buffer...60ml GT Buffer...60ml W1 Buffer... 50ml Wash Buffer (Concentrated)*...25ml Elution Buffer...30ml GD Maxi Column Set...10 Sets (Provided with 50ml Centrifuge Tube) Cat.No. YGBM25 25 maxi preps / kit RBC Lysis Buffer...500ml x2 GB Buffer...150ml GT Buffer...150ml W1 Buffer...130ml Wash Buffer (Concentrated) *...40ml Elution Buffer...60ml GD Maxi Column Set...25 Sets (Provided with 50ml Centrifuge Tube) Sample (Protocols Included): Fresh/ Frozen Blood, Bacteria Average Yields: Fresh Blood : 140μg. Frozen Blood : 75μg. * Add 4 times volume of ethanol(96-100%) to Wash Buffer prior to initial use. Additional Requirement: Microcentrifuge Tube, Ethanol (96-100%), RNase A (10 mg/ml). For best result, Swing-Bucket Centrifuge is strongly recommended for following protocols. 13 Genomic DNA Extraction Kit- Maxi - Blood/Bacteria/Cultured Cells

19 Description Genomic DNA Extraction Maxi Kit (Blood) provides a fast and economical method for purification of total DNA (including genomic, mitochondrial and viral DNA) from whole blood, plasma, serum, buffy coat, other body fluids, lymphocytes and cultured cells. This kit is also ideal for bacteria and cultured cells. The blood protocol utilises RBC Lysis Buffer, GB 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 Extraction Kit- Maxi - Blood/Bacteria/Cultured Cells 14

20 Fresh Blood Protocol (<10ml) RBC Lysis 1. Collect fresh blood in EDTA-Na 2 treated collection tubes (or other anticoagulant mixtures). 2. Add up to 10 ml of blood to a 50 ml Centrifuge Tube. 3. Add 3 times the sample volume of RBC Lysis Buffer and mix by inversion. Do not vortex. 4. Incubate the tube for 10 minutes at room temperature. 5. Centrifuge for 5 minutes at 4,000 x g and discard the supernatant. 6. Add 1 ml RBC Lysis Buffer to resuspend the pellet. Cell Lysis 7. Add 5 ml GB Buffer to the tube and mix by vortexing. 8. Incubate the mixture in a 60 C water bath for 10 minutes until the sample lysate is clear. During incubation, invert the tube every 3 minutes. At this time, preheat required Elution Buffer (2 ml per sample) in a 60 C water bath (For DNA Elution). Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform the optional step. a. After 70 C incubation, add 50μl of RNase A (10mg/ml, provided by user) to sample lysate and vortex to mix sample. b. Incubate at room temperature for 5 minutes. 15 Genomic DNA Extraction Kit- Maxi - Blood Protocol

21 DNA Binding 9. Add 5 ml of ethanol (96~100%) to the sample lysate and mix immediately by vortexing for 10 seconds. If precipitate appears, break up by pipetting. 10. Place a GD Maxi Column on a 50 ml Centrifuge Tube. 11. Apply all the mixture (including any precipitate) from the previous step to the GD Maxi Column. 12. Close the cap and centrifuge at 4,000 x g for 5 minutes. 13. Discard the flow-through and return the GD Maxi Column to the 50ml Centrifuge Tube. Wash 14. Add 4 ml of W1 Buffer into the column. 15. Centrifuge at 4,000 x g for 3 minutes. 16. Discard the flow-through and return the GD Maxi Column to the 50 ml Centrifuge Tube. 17. Add 6 ml of Wash Buffer (ethanol added) into the column. 18. Centrifuge at 4,000 x g for 3 minutes. 19. Discard the flow-through and return the GD Maxi Column to the 50 ml Centrifuge Tube. 20. Centrifuge at full speed for 10 minutes to dry the column matrix. DNA Elution Standard elution volume is 2 ml. If higher DNA yield is required, repeat the DNA Elution Step to increase DNA recovery and the total elution volume to about 4 ml. 21. Transfer dried GD Maxi Column into a clean 50 ml centrifuge tube (not provided). 22. Add 2 ml of preheated Elution Buffer to the center of the column matrix. 23. Allow to stand for 3-5 minutes until Elution Buffer is absorbed by the matrix. 24. Centrifuge at 4,000 x g for 2 minutes to elute purified DNA. Genomic DNA Extraction Kit- Maxi - Blood Protocol 16

22 Frozen Blood Protocol (<5ml) Sample (Protocols Included): 5 ml, Yield: 70μg, Elution Volume: 1.5 ml, Average Length: 20-30kb, Operation Time: 60 mins. Add 4 times volume of ethanol(96-100%) to Wash Buffer prior to initial use. Additional Requirement: 50 ml centrifuge tube, Proteinase K (20 mg/ml), Ethanol (96-100%), RNase A (10mg/ml) Cell Lysis 1. Apply up to 5 ml of blood to a 50 ml centrifuge tube. 2. Add 2.5 ml GB Buffer and 200 μl Proteinase K (20 mg/ml) (not provided) to the tube and mix by vortexing. 3. Incubate the mixture in a 60 C water bath for 20 minutes until the sample lysate is clear. During incubation, invert the tube every 3-5 minutes. At this time, preheat required Elution Buffer (1.5 ml per sample) in a 60 C water bath (For DNA Elution ). Optional Step: RNA Degradation If RNA free genomic DNA is required, perform this optional step a. Add 50μl of RNase A (10mg/ml, not provided) to sample lysate and mix by vortexing. b. Allow to stand at room temperature for 10 minutes.b.allow to stand at room temperature for 10 minutes. DNA Binding 4. Add 5 ml of ethanol (96~100%) to the sample lysate and mix immediately by vortexing for 10 seconds. If precipitate appears, break up by pipetting. 5. Place a GD Maxi Column in a 50 ml centrifuge tube. 6. Apply all the mixture (including any precipitate) from the previous step to the GD-Maxi Column. 7. Close the cap and centrifuge at 4,000 x g for 5 minutes. 8. Discard the flow-through and return the GD Maxi Column to the 50ml Centrifuge Tube. 17 Genomic DNA Extraction Kit- Maxi - Blood Protocol

23 Wash 9. Add 4 ml of W1 Buffer to the column. 10. Centrifuge at 4,000 x g for 3 minutes. 11. Discard the flow-through and return the GD Maxi Column to the 50 ml Centrifuge Tube. 12. Add 6 ml of Wash Buffer (ethanol added) into the column. 13. Centrifuge at 4,000 x g for 3 minutes. 14. Discard the flow-through and return the GD Maxi Column to the 50 ml Centrifuge Tube. 15. Centrifuge at full speed for 10 minutes to dry the column matrix. DNA Elution Standard elution volume is 1.5 ml. If higher DNA yield is required, repeat the DNA Elution Step to increase DNA recovery and the total elution volume to about 3 ml. 16. Transfer dried GD Maxi Column into a clean 50 ml centrifuge tube (not provided). 17. Add 1.5 ml of preheated Elution Buffer to the center of the column matrix. 18. Allow to stand for 3-5 minutes until Elution Buffer is absorbed by the matrix. 19. Centrifuge at 4,000 x g for 2 minutes to elute purified DNA. Genomic DNA Extraction Kit- Maxi - Blood Protocol 18

24 Troubleshooting Problem Column clogged Possible Reason/Solution Overloaded column with sample Reduce sample volume or separate into multiple tubes. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. Low yield Incorrect DNA Elution Step Ensure that Elution Buffer was added and absorbed to the centre of GD-Maxi Column matrix. Incomplete DNA Elution Elute twice to increase yield Eluted DNA does not perform well in downstream applications Residual ethanol contamination Following the wash step, dry GD-Maxi Column with additional centrifugation at full speed for 10 minutes or incubation at 60 C for 10 minutes. RNA Contamination Perform optional RNA degradation step. Genomic DNA was degraded Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. 19 Genomic DNA Extraction Kit- Maxi - Blood/Bacteria/Cultured Cells

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26 Genomic DNA Extraction Kit Mini Tissue Cat.No. YGT50//YGT100 Kit Contents Cat.No. YGT50 50 mini preps / kit GB Buffer...15ml GT Buffer...15ml W1 Buffer... 25ml Wash Buffer (Concentrated)*...25ml Elution Buffer...30ml Proteinase K **...11 mg GD Column...50 pcs 2 ml Collection Tube...50 pcs Micropestles...50pcs Cat.No. YGT mini preps / kit GB Buffer...30ml GT Buffer...30ml W1 Buffer...50ml Wash Buffer (Concentrated) *...25ml Elution Buffer...30ml Proteinase K **...11 mg x 2 GD Column pcs 2 ml Collection Tube pcs Micropestles...100pcs Sample (Protocols Included): 20mg of tissue, 0.5 cm of mouse tail Average Yields: Tissue, Paraffin-Embedded Tissue, Buccal Swab * Add 4 times volume of ethanol (96-100%) to Wash Buffer prior to initial use. ** Add 1.1 ml ddh 2 0 to the tube and mix by vortexing. Store prepared Proteinase K (10mg/ml) at 4 C. For long term storage,aliquot and store at -20 C. 21 Genomic DNA Extraction Kit - Mini - Tissue

27 Description Genomic DNA Extraction Mini Kit (Tissue) has been designed for purification of total DNA (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 for lysis. The method uses proteinase K and a chaotropic salt, guanidine hydrochloride to lysis cells and degrade protein. DNA in chaotropic salt 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. Quality Control The quality of Genomic DNA Extraction Mini Kit (Tissue) is tested on a lot-to-lot basis. The kits are tested by isolation of genomic DNA from 10mg of mouse liver. Purified DNA is quantified with a spectrophotometer. Genomic DNA yield is more than 10μg with A260/A280 ratio 1.7 to 1.9. Applications PCR, Southern Blotting, RADP/ AFLP Reference Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615. Note * For research use only. Not for use in diagnostic or therapeutic procedures. * Buffer contains guanidine hydrochloride which is harmful and can act as an irritant. During operation, always wear a lab coat, disposable gloves and protective goggles. Genomic DNA Extraction Kit - Mini - Tissue 22

28 60 C 30min 70 C 20min GT Buffer Proteinase K GB Buffer DNA Binding Wash Step Elution Step Purified DNA 23 Genomic DNA Extraction Kit - Mini - Tissue Protocol

29 Tissue Protocol Add 1.1ml ddh 2 O to a Proteinase K (11mg) tube, vortex to dissolve. Store prepared Proteinase K at 4 C. Add 100ml ethanol (96-100%) to Wash Buffer before initial use. Additional Requirement: Microcentrifuge tube, Ethanol (96-100%), RNase A (10mg/ml) Tissue Dissociation 1. Cut up 20mg of animal tissue (or 0.5cm of mouse 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 10mg. 2. Use provided micropestle to grind the tissue to a pulp. 3. Add 200μl GT Buffer into the tube and continue to homogenize the sample tissue with grinding. Cell Lysis 4. Add 20μl Proteinase K (10mg/ml) to the sample mixture and vortex to mix. Incubate at 60 C for 30 minutes to lyse the sample. During incubation, invert the tube every 5 min. 5. Add 200μl GB Buffer and vortex for 5 seconds to mix sample. 6. Incubate at 70 C for 20 minutes until the sample lysate is clear. During incubation, invert the tube every 5 min. At this time, preheat required Elution Buffer (200μl per sample) in a 70 C water bath (For DNA Elution). *If there is insoluble material present following incubation, centrifuge for 2 minutes at full speed (13,000 rpm) and transfer the supernatant to a new microcentrifuge tube (not provided). Genomic DNA Extraction Kit - Mini - Tissue Protocol 24

30 Optional Step: RNA Degradation If RNA-free genomic DNA is required, perform this optional step. a. Following 70 C incubation, add 5μl of RNase A (10mg/ml) (not provided) to sample lysate and vortex to mix. b. Incubate at room temperature for 5 minutes. DNA Binding 7. Add 200μl of ethanol (96-100%) to the sample lysate and immediately vortex for 10 seconds to mix. If precipitate appears, break up by pipetting. 8. Place a GD Column in a 2ml Collection Tube. Apply the total mixture (including any precipitate) from previous step to the GD Column. 9. Centrifuge at 10,000 x g (13,000 rpm) for 2 minutes. Discard the flow-through and return the GD column to the 2ml Collection Tube. Wash 10. Add 400μl of W1 Buffer to the GD Column. Centrifuge at10,000 x g (13,000 rpm) for 30 seconds. 11. Discard the flow-through and return the GD Column to the 2ml Collection Tube. 12. Add 600μl of Wash Buffer (ethanol added) in the GD Column. 13. Centrifuge at 10,000 x g (13,000 rpm) for 30 seconds. 14. Discard the flow-through and return the GD Columnto the 2ml Collection Tube. 15. Centrifuge at 10,000 x g (13,000 rpm) for 3 minutes to dry the column matrix. 25 Genomic DNA Extraction Kit - Mini - Tissue Protocol

31 DNA Elution Standard elution volume is 100μl. If less sample volume is used, reduce the elution volume (15-50μl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution Step to increase DNA recovery and the total elution volume to about 200μl. 16. Transfer dried GD Column to a clean 1.5 ml microcentrifuge tube (not provided). 17. Add 100μl of preheated Elution Buffer to the centre of the column matrix. 18. Allow to stand for 2 minutes until Elution Buffer is absorbed by the matrix. 19. Centrifuge at 10,000x g (13,000 rpm) for 30 seconds to elute purified DNA. Genomic DNA Extraction Kit - Mini - Tissue Protocol 26

32 For Paraffin-embedded Tissue Additional Requirement: Xylene, Microcentrifuge Tube, Ethanol (96-100%) Sample Preparation 1. Slice small sections (up to 25mg) from blocks of paraffin-embedded tissue and transfer to a microcentrifuge tube. 2. Add 1ml xylene to each tube. Vortex vigorously and incubate at room temperature for about 10 min. Vortex occasionally during incubation step. 3. Centrifuge at 10,000 x g (13,000 rpm) for 3 min. Remove supernatant by pipetting. 4. Add 1 ml ethanol to wash sample pellet and mix by inverting. 5. Centrifuge at 10,000 x g (13,000 rpm) for 3 min. Remove supernatant by pipetting. Repeat the Wash Step. 6. Open tube and incubate at 37 C for 15 minutes to evaporate the residual ethanol. 7. Add 200μl GT Buffer to suspend the sample then Proceed to Cell Lysis Step 4 of Tissue protocol (page 24). 27 Genomic DNA Extraction Kit - Mini - Tissue Protocol

33 For Buccal Swab Additional Requirement: Swab: cotton, DACRON or C.E.P. swabs; PBS; Microcentrifuge Tube; Ethanol (96-100%) 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 for 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 add 500μl PBS. Lysis 3. Add 500μl GB Buffer and 20μl Proteinase K (10mg/ml) to the sample. Vortex for 5 seconds to mix sample. 4. Incubate at 70 C for 20 minutes. During incubation, invert the tube every 5 minutes. At this time, preheat required Elution Buffer (200μl per sample) in a 70 C water bath to be used in Elution Step.) DNA Binding 5. Add 500μl of ethanol (96-100%) to the sample lysate and vortex immediately to mix. 6. Place a GD Column on a 2 ml Collection Tube. 7. Apply 700μl of the mixture from the previous step to the GD Column. 8. Close the cap and centrifuge at 10,000 x g (13,000 rpm) for 1 minute.discard the flow-through and return the GD column to the 2ml Collection Tube. 9. Repeat DNA Binding Step by applying the remaining mixture to GD Column. 10. Proceed to Wash Step 10 of Tissue Protocol (page 25). Genomic DNA Extraction Kit - Mini - Tissue Protocol 28

34 Troubleshooting Problem Column clogged Possible Reason/Solution Overloaded column with sample Reduce sample volume or separate into multiple tubes. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. Low yield Incorrect DNA Elution Step Ensure that Elution Buffer was added and absorbed to the centre of GD Column matrix. Incomplete DNA Elution Elute twice to increase yield Eluted DNA does not perform well in downstream applications Residual ethanol contamination Following the wash step, dry GD Column with additional centrifugation at full speed for 5 minutes or incubation at 60ºC for 5 minutes. RNA Contamination Perform optional RNA degradation step. Protein Contamination Reduce the sample amount After DNA Binding Step, apply 400 μl W1 Buffer to GD Column and centrifuge at 13,000 rpm for 30 seconds. Proceed with Wash Step. Genomic DNA was degraded Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. 29 Genomic DNA Extraction Kit - Mini - Tissue

35 M RBC Bioscience Labs Comparison: Genomic DNA Mini Kit (Tissue) vs Brand Q Genomic DNA Extraction Kit 30mg/sample: Lane 1 and 2 (Liver), Lane 3 and 4 (Kidney), Lane 5 and 6 (Spleen) Lane 1,3,5 -Brand Q Lane 2,4,6 -RBC Real Genomics RBC Bioscience Labs Genomic DNA Extraction Kit - Mini - Tissue 30

36 Genomic DNA Extraction Kit Mini Plant Cat.No. YGP50//YGP100 Kit Contents Cat.No. YGP50 50 mini preps / kit GP1 Buffer...25ml GPX1 Buffer...25ml GP2 Buffer... 6ml GP3 Buffer*... 15ml W1 Buffer... 25ml Wash Buffer (Concentrated)**...25ml Elution Buffer...30ml RNase A (10mg/ml)...275μl Filter Column...50 pcs GD Column...50 pcs 2 ml Collection Tube...100pcs Cat.No. YGP mini preps / kit GP1 Buffer...50ml GPX1 Buffer...50ml GP2 Buffer... 15ml GP3 Buffer*... 30ml W1 Buffer... 50ml Wash Buffer (Concentrated)**...25ml Elution Buffer...30ml RNase A (10mg/ml)...550μl Filter Column pcs GD Column pcs 2 ml Collection Tube...200pcs Sample (Protocols Included): 1g of plant tissue Yield: 300μg Operation time: < 60 mins Elution Volume: 50μl * Add 2 times volume isopropanol to GP3 Buffer prior to initial use. ** Add 4 times volume ethanol (96-100%) to Wash Buffer prior to initial use. Additional Requirement : 15ml, 50ml Centrifuge Tube. 31 Genomic DNA Extraction Kit - Mini - Plant

37 Description The Plant Genomic DNA Extraction Kit (Duo Buffer System) 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 a chaotropic salt, the genomic DNA in the lysate binds to the glass fiber matrix in the spin column. The contaminants 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 DNA phenol extraction and alcohol precipitation. The entire procedure can be completed in 60 minutes. Quality Control The quality of Genomic DNA Extraction Mini Kit (Tissue) is tested on a lot-to-lot basis. The kits are tested by isolation of genomic DNA from 10mg of mouse liver. Purified DNA is quantified with a spectrophotometer. Genomic DNA yield is more than 10μg with A260/A280 ratio 1.7 to 1.9. Applications PCR, Southern Blotting, RADP/ AFLP Reference Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615. Note * For research use only. Not for use in diagnostic or therapeutic procedures. * Buffer contains guanidine hydrochloride which is harmful and can act as an irritant. During operation, always wear a lab coat, disposable gloves and protective goggles. Genomic DNA Extraction Kit - Mini - Plant 32

38 65 C 10min Tissue Dissociation GP1/GPX1 Buffer GP2 Buffer Incubate on Ice GP3 Buffer DNA Binding Wash Step Elution Step Purified DNA Additional Requirements: Liquid Nitrogen, Mortar, 1.5 ml microcentrifuge tube, Ethanol (96-100%), Isopropanol. 33 Genomic DNA Extraction Kit - Mini - Plant

39 Protocol Modification (Duo Buffer System) Plant species are extremely diverse in their metabolic components. Large amounts of polysaccharides, carbohydrates, lipids, poly-phenols and proteins may be distributed throughout the plant tissue. These compounds often interfere with DNA binding and extraction. Due to this characteristic of plants, we offer two lysis buffers for optimum performance according to different plant samples. GP1: The standard protocol utilizes GP1 buffer for plant sample lysis. This buffer system is suitable for most common plant species, to ensure high quality and high yield of DNA. GPX1: An alternative buffer, GPX1, is also included with this kit. The detergent present in this buffer is more effective in dispersing plant samples with large amounts of polysaccharide. For most of plant species, both buffers give similar results. Researchers may try one buffer system first or both in parallel. Genomic DNA Extraction Kit - Mini - Plant 34

40 Plant Protocol Tissue Dissociation 1. Cut off 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). Lysis 4. Add 400 μl GP1 Buffer (or GPX1) and 5 μl RNase A (10mg/ml) into the sample tube and mix by vortexing. Do not mix GP1 (GPX1) Buffer with RNase A before use. 5. Incubate at 65 C for 10 minutes. During incubation, invert the tube every 5 minutes. At the same time, preheat required Elution Buffer (200 μl per sample) at 65 C. 6. Add 100μl GP2 Buffer and mix by vortexing. 7. Incubate on ice for 3 minutes. Place a Filter Column into a 2 ml Collection Tube and apply the entire lysate from previous step to the Filter Column. 8. Centrifuge for 3 minutes at full speed (13,000 rpm). 9. Discard the Filter Column and carefully transfer clarified supernatant in the Collection Tube to a new microcentrifuge tube ( not provided). DNA Binding 10. Add 1.5 times volume of GP3 Buffer (isopropanol added) to the cleared lysate and mix immediately by vortexing for 5 seconds. For example, add 750 μl GP3 Buffer to 500 μl lysate. 11. Place a GD Column into a 2 ml Collection Tube. 12. Apply 700 μl of the mixture (including any precipitation) from previous step to the GD Column. 13. Centrifuge at full speed (13,000 rpm) for 2 minutes. 14. Discard flow-through in Collection Tube and apply remaining mixture to GD Column. 15. Repeat step12-14 again. Apply the remaining mixture to the same GD Column 35 Genomic DNA Extraction Kit - Mini - Plant Protocol

41 16. Discard flow-through in Collection Tube. Wash 17. Add 400 μl of W1 Buffer to the GD Column. 18. Centrifuge at full speed (13,000 rpm) for 30 seconds. 19. Discard the flow-through and place the GD Column back in the Collection Tube. 20. Add 600 μl of Wash Buffer (ethanol added) to the GD Column. 21. Centrifuge at full speed (13,000 rpm) for 30 seconds 22. Discard the flow-through and place the GD Column back in the Collection Tube 23. Centrifuge at full speed for 3 minutes to dry out column matrix. Optional Step: RNA Degradation If a few pigments remain on the column matrix, perform this procedure. a. After Wash Steps are completed, add 400 μl of ethanol (96-100%) in the GD Column. b. Centrifuge at full speed (13,000 rpm) for 30 seconds c. Discard the flow-through and place the GD Column back in the Collection Tube d. Centrifuge at full speed for 3 minutes to dry out column matrix.following 70 C incubation, add 5μl of RNase A (10mg/ml) (not provided) to sample lysate and vortex to mix. e. Incubate at room temperature for 5 minutes. DNA Elution Standard elution volume is 100μl. If less sample to be used, reduce the elution volume (30-50μl) to increase DNA concentration. If higher DNA yield required, repeat the DNA Elution Step to increase DNA recovery and the total elution volume to about 200μl. 24. Transfer dried GD Column to a clean 1.5 ml microcentrifuge tube ( not provided) 25. Add 100 μl of preheated Elution Buffer to the center of the column matrix. 26. Allow to stand for 3-5 minutes until Elution Buffer is absorbed by the matrix. 27. Centrifuge at full speed (13,000 rpm) for 30 seconds to elute purified DNA. Genomic DNA Extraction Kit - Mini - Plant Protocol 36

42 Troubleshooting Problem Column clogged Possible Reason/Solution Overloaded column with sample Reduce sample volume or separate into multiple tubes. Precipitate was formed at DNA Binding Step Reduce the sample material. Prior to loading the column, break up precipitate in ethanol-added lysate. Low yield Incorrect DNA Elution Step Ensure that Elution Buffer was added and absorbed to the centre of GD Column matrix. Incomplete DNA Elution Elute twice to increase yield Eluted DNA does not perform well in downstream applications Residual ethanol contamination Following the wash step, dry GD Column with additional centrifugation at full speed for 5 minutes or incubation at 60ºC for 5 minutes. RNA Contamination Perform optional RNA degradation step. Protein Contamination Reduce the sample amount After DNA Binding Step, apply 400 μl W1 Buffer to GD Column and centrifuge at 13,000 rpm for 30 seconds. Proceed with Wash Step. Genomic DNA was degraded Use fresh tissue sample; prolonged storage may result in fragmentation of genomic DNA. 37 Genomic DNA Extraction Kit - Mini - Plant

43 Fig 1. Agarose gel (1.0%) analysis of DNA isolated from the indicated young leaves with the RBC Plant Genomic DNA extraction system. (100 mg sample, loading 4 ml) Lane 1: Marker Lane 2: Cinnamommun camohora (Camphor tree) Lane 3: Pisum sativum (Pea Sprout) Lane 4: Arabidopsis thaliana (Arabidopsis) Lane 5: Oryza sativa (Rice) Lane 6: Ipomoea batatas. ( Sweet potato vine ) Lane 7: Rhizoma dioscoreae (Chinese Yam) Lane 8: Populus tremula (aspen) Lane 9: Flammulina velutipes Lane 10: Oxalis comiculats.(fourleaf clover) Fig 2. Compare yield between Brand Q and RBC Real Genomics (100 mg sample, loading 8 ml) Lane 1: Brand Q Arabidopsis thaliana (Arabidopsis) Lane 2: RBC Arabidopsis thaliana (Arabidopsis) Lane 3:BRAND Q Pisum sativum (Pea sprout) Lane 4: RBC Pisum sativum (Pea sprout) Lane 5: BRAND Q Populus tremula (Aspen) Lane 6: RBC Populus tremula (Aspen) Lane 7: BRAND Q Bambusa lodhamii Munro (Bamboo) Lane 8: RBC Bambusa lodhamii Munro (Bamboo) Genomic DNA Extraction Kit - Mini - Plant 38

44 Genomic DNA Extraction Kit Maxi Plant Cat.No. YGPM10//YGPM25 Kit Contents Cat.No. YGPM10 10 maxi preps / kit GP1 Buffer...50ml GPX1 Buffer...50ml GP2 Buffer...15ml GP3 Buffer*... 30ml W1 Buffer... 50ml Wash Buffer (Concentrated)**...25ml Elution Buffer...30ml RNase A (10mg/ml)...550μl 50ml Centrifuge...10pcs Filter Maxi Column...10 pcs GD Maxi Column Set...10 Sets (Provided with 50ml Centrifuge Tube) Cat.No. YGPM25 25 maxi preps / kit GP1 Buffer...125ml GPX1 Buffer...125ml GP2 Buffer... 30ml GP3 Buffer*... 70ml W1 Buffer...130ml Wash Buffer (Concentrated)**...40ml Elution Buffer...60ml RNase A (10mg/ml)...650μl X2 50ml Centrifuge...25pcs Filter Maxi Column...25pcs GD Maxi Column...25 pcs Sample (Protocols Included): 1g of plant tissue Yield: 300μg Operation time: < 60 mins Elution Volume: 1000μl * Add 2 times volume isopropanol to GP3 Buffer prior to initial use. ** Add 4 times volume ethanol (96-100%) to Wash Buffer prior to initial use. Additional Requirement : 15ml, 50ml Centrifuge Tube. 39 Genomic DNA Extraction Kit - Maxi - Plant

45 Description Plant Genomic DNA Maxi Kits provide a fast and simple method to isolate total DNA (genomic DNA,mitochondrial and chloroplast) from plant tissue and cells. Samples are homogenized under liquid nitrogen followed by lysis buffer incubation. The lysate is treated with RNase A to degrade RNA and spun through a filtercolumn to remove cell debris and salt in the solution. In the presence of binding buffer and chaotropic salt, the genomic DNA in the lysate binds to the glass fiber matrix in the spin column. The contaminants are washed with an ethanol based wash buffer and the purified genomic DNA is eluted by low salt elution buffer or water. The purified genomic DNA is ready for PCR, real-time PCR, Southern blotting and RFLP. Quality Control The quality of the Plant Genomic DNA kits are tested on a lot-to-lot basis. The kits are tested by isolation of genomic DNA from 500 mg of young leaves. Caution The above components contain irritants. During the extraction process always wear a lab coat, disposable gloves and protective goggles. Protocol Modification (Duo Buffer System) Plant species are extremely diverse in their metabolic components. Large amounts of polysaccharides,carbohydrates, lipids, polyphenols and proteins may be distributed throughout the plant tissue. These compounds often interfere with DNA binding and extraction. This characteristic of plants means we offer two lysis buffers for optimum performance according to plant sample type. GP1: The standard protocol utilizes GP1 buffer for lysis of plant samples. This buffer system is suitable for most common plant species, ensuring high quality and high yields of DNA. GPX1: An alternative buffer, GPX1, is also included with this kit. The detergent present in this buffer is more effective in dispersing plant samples with large amounts of polysaccharide. For the majority of plant species both buffers will give similar results. The researcher may try one buffer system first or both in parallel. Genomic DNA Extraction Kit - Maxi - Plant 40

46 Plant Protocol Tissue Dissociation 1. Cut off 0.5 g (up to 1g) of fresh or frozen plant tissue or 50 mg (up to 100 mg) of dried sample. 2. Grind the sample under liquid nitrogen to a fine powder. Transfer it into a 15 ml centrifuge tube (not provided). It is possible to render some plant samples without the aid of liquid nitrogen. Lysis 3. Add 4 ml GP1 Buffer (or GPX1 Buffer) and 50 μl RNase A (10 mg/ ml) to the sample tube and mix by vortexing. (Do not mix GP1 Buffer and RNase A before use.) 4. Incubate at 65 C for 20 minutes. During incubation, invert the tube every 5 minutes. At the same time, preheat required Elution Buffer (2 ml per sample) at 65 C. 5. Add 1 ml GP2 Buffer and mix by vortexing. 6. Incubate on ice for 5 minutes. 7. Place a Filter Maxi Column in a 50 ml Centrifuge Tube. 8. Apply the mixture from previous step to the Filter Maxi Column. Centrifuge at 4,000 xg for 5 min. 9. Discard the Filter Maxi Column and carefully transfer clarified supernatant in collection tube to a new 50 ml Centrifuge Tube (not provided). DNA Binding 10. Add 1.5 times volume of GP3 Buffer (isopropanol added) to the cleared lysate and mix immediately by vortexing for 10 seconds. For example, add 7.5 ml GP3 Buffer to 5 ml of lysate. 11. Place a GD Maxi Column in a 50 ml Centrifuge Tube. 12. Apply the mixture (including any precipitate) from previous step to the GD Maxi Column. 13. Centrifuge at 4,000 x g for 5 minutes. 14. Discard the flow-through and place the GD Maxi Column back in the collection t ube. 41 Genomic DNA Extraction Kit - Maxi - Plant Protocol

47 Wash 15. Add 4 ml of W1 Buffer into the column. 16. Centrifuge at 4,000 x g for 3 minutes. 17. Discard the flow-through and place the GD Maxi Column back in the collection tube. 18. Add 6 ml of Wash Buffer (ethanol added) into the column. 19. Centrifuge at 4,000 x g for 3 minutes. 20. Discard the flow-through and place the GD-Maxi Column back in the collection tube. 21. Centrifuge at 4,000 x g for 10 minutes to dry the column matrix. DNA Elution Standard elution volume is 1 ml. If less sample is used, reduce the elution volume ( μl) to increase DNA concentration. If higher DNA yield is required, repeat the DNA Elution step to increase DNA recovery by bringing the total elution volume to about 2 ml. 22. Transfer dried GD-Maxi into a clean 50 ml centrifuge tube (not provided). 23. Add 1 ml of preheated Elution Buffer to the center of the column matrix. 24. Allow to stand for 5 minutes until Elution Buffer is absorbed by the matrix. 25. Centrifuge at 4,000 x g for 3 minutes to elute purified DNA. Genomic DNA Extraction Kit - Maxi - Plant Protocol 42

48 RBC Real Genimic DNA Extraction Kit Product Information DNA Fragment Viral Plasmid Genomic Item / Cat.No. Genomic DNA Extraction Kit Mini (Blood/Bacteria/Cultured Cells) YGB50,YGB100,YGB300 Genomic DNA Extraction Kit Maxi (Blood/Bacteria/Cultured Cells) YGBM10,YGBM25 Genomic DNA Extraction Kit Mini (Tissue) YGT50,YGT100 Genomic DNA Extraction Kit Mini (Plant) YGP50,YGP100 Genomic DNA Extraction Kit Maxi (Plant) YGPM10,YGPM25 Genomic DNA Extraction Kit (Cell Lysus Buffer) YGE well Genomic DNA Extraction Kit RBP96B-2,RBP96B-4,RBP96B-10 YPD100 YPD300 YPM10 HiYield Plasmid Kit Mini HiYield Plasmid Kit Mini (High Throughput Applications) Fastlon Plasmid Kit Maxi (Ion Exchange,High Yield/Purity) YPI25 Fastlon Plasmid Kit Maxi (Ion Exchange,High Yield/Purity) 96-well Plasmid Kit YPD96B-2,YPD96B-4,YPD96B-10 YVN50 Viral Nucleic Acid Extraction Kit YVN100 Viral Nucleic Acid Extraction Kit 96-well Viral Nucleic Acid Extraction Kit YVN96B-2,YVN96B-4,YVN96B-10 HiYield Gel / PCR DNA Fragments Extraction Kit YDF100,YDF300 G50 Dye Terminator Removal Kit (For Sequencing Reactions) YGC50 G25 Oligo Clean Up Kit YGC25 96-well PCR Clean Up Kit YDF96B-2,YDF96B-4,YDF96B-10 Specification (RBC Lysis,GB,GT,W1,Wash,Elution) Buffer, Proteinase K 50 spin preps/kit, 100 spin preps/kit, 300 spin preps/kit (RBC Lysis, GB, GT, W1, Wash, Elution) Buffer 10 preps/kit,25 preps/kit (GT, GB, W1, Wash, Elution) Buffer, Proteinase K 50 preps/kit, 100preps/kit (GP1, GPX1, GP2, GP3, W1, Wash, Elution) Buffer, RNase A 50 preps/kit, 100preps/kit (GP1, GPX1, GP2, GP3, W1, Wash, Elution) Buffer, RNase A 10preps/kit, 25preps/kit (RBC Lysis, Cell Lysis) Buffer 100ml/kit (GB, W1, Wash, Elution) Buffer, Proteinase K 2 Binding Plates/kit,4 Binding Plates/kit,10 Binding Plates/kit 100 preps / kit (PD1, PD2, PD3, W1, Wash, Elution) Buffer, RNase A 300 preps / kit (PD1, PD2, PD3, W1, Wash, Elution) Buffer, RNase A 10 preps / kit (PM1, PM2, PM3, PEQ, PW, PEL) Buffer, RNaseA 25 preps / kit (PM1, PM2, PM3, PEQ, PW, PEL) Buffer, RNaseA (PDA1,PDA2,PDA3,W1,Wash, Elution) Buffer, RNase A, Sealing Film 2 Binding Plates/kit,4 Binding Plates/kit,10 Binding Plates/kit 50 preps / kit (VB,W1,R-Wash) Buffer, Carrier RNA, RNase-Free Water 100 preps / kit (VB,W1,R-Wash) Buffer, RNase-Free Water, Carrier RNA, Sealing Film (VB,W1,R-Wash) Buffer, RNase-Free Water, Carrier RNA, Sealing Film 2 Binding Plates/kit,4 Binding Plates/kit,10 Binding Plates/kit (DF, Wash, Elution) Buffer 100 preps/kit,300 preps/kit G50 Columns(sephadex based) 50pcs/kit G25 Columns (sephadex based) 50pcs/kit (Binding, Wash, Elution) Buffer, Sealing Film 2 plates, 4 plates, 10 plates/kit 43

49 RBC Real Genimic RNA Extraction Kit Product Information Item / Cat.No. Specification Total RNA Viral RNA Total RNA Extraction Kit Mini (Blood/Bacteria/Cultured Cells) YRB50,YRB100,YRB300 Total RNA Extraction Kit Maxi (Blood/Bacteria/Cultured Cells) YRBM10,YRBM25 Total RNA Extraction Kit Mini (Tissue) YRT50,YRT100 Total RNA Extraction Kit Mini (Plant) YRP50,YRP100 Total RNA Extraction Kit Maxi (Plant) YRPM10,YRPM25 Viral Nucleic Acid Extraction Kit YVN50,YVN well Viral Nucleic Acid Extraction Kit YVN96B-2,YVN96B-4,YVN96B-10 (RBC Lysis, RB, RT, R-W1, R-Wash, RNase-Free Water) Buffer 50 spin preps/kit, 100 spin preps/kit, 300 spin preps/kit (RBC Lysis, RB, R-W1, R-Wash, RNase-Free Water) Buffer 10 preps/kit,25 preps/kit (RB, R-W1, R-Wash, RNase-Free Water) Buffer 50 preps/kit, 100preps/kit (RB, PRB, R-W1, R-Wash, RNase-Free Water) Buffer 50 preps/kit, 100preps/kit (RB, PRB, R-W1, R-Wash, RNase-Free Water) Buffer 10 preps/kit,25 preps/kit (VB,R-W1, R-Wash) Buffer, Carrier RNA, RNase-Free Water 50 spin preps/kit, 100 spin preps/kit (VB,R-W1, R-Wash) Buffer, RNase-Free Water, Carrier RNA, Sealing Film 2 plates, 4 plates, 10 plates/kit RBC Real Genimic Purification Enzymes Information Cat.No. YPK10 RN050 RN130 YLY20 Specification Protelnase K RNase A RNase A Lysozyme 44

50 Automated Nucleic Acid Extractor MagCore HF16

51

52 Easy To Use Simple Steps 1 Install all necessary accessories and apply your specimen to MagCore 2 Input Catridge Code to run protocol Note:For research use only. Not for use in diagnostic or therapeutic procedures. 3 30min~45min to complete!! Processing Capacity More higher processing capacity to handle up to 16 samples synchronised.

53 To avoid cross contamination UV Sterilization UV button is designed to turn on UV Lamp while your MagCore HF16 needs to be sterilized after runing contagious samples. Independent Channel Pipetting system and dispossable accessaries for each independent channel provide corss-contamination free to MagCore HF16. Built-in programs With pre-programmed protocols built-in, MagCore is available for a diverse range of specimen. All programs are controlled by a different 3-digital protocol code, simply kiy-in the code to run desired protocol. MagCore is equipped with RS232 port for software upgrade, simply download from RBC website for free updation.

54 Execellent Accessories Design Cartridge Design MagCore reagent cartridges come pre-sealed incorporating all reagents needed for the purification process. No additional reagents are required and no handling is necessary beyond placing inside the instrument. Pre-filled and sealed buffer cartridge and automated pierceing step, it eliminates possible contaminations or buffer spout. Patented Heating Well and Separation Well in the cartridge provide strong circulate force for Binding/Washing steps and give high purity in final elutant. Each cartridge has 14 positions available with 10 sealed wells and two optional heating positions as illustrated in the diagram below. Magnetic beads separation well 2 Magnetic beads separation well 1 ProteinaseK Lysis Buffer Binding Buffer MagCore Beads Wash 1 Buffer Wash 2 Buffer Wash 3 Buffer Elution Buffer Heating well 2 Heating well 1 Sample vial Elutant The MagCore beads are pre-allocated into each cartridge in Well 4. Mixing of the beads, reagents and sample is achieved by the liquid handling action of the instrument. Separation is achieved by magnetic attraction and liquid handling.

55 Tip Design The unique design of Tip-end with cross notch, provides prcise buffer volume at pipetting steps.