FavorPrep Soil RNA Isolation Mini Kit

Transcription

1 FavorPrep Soil RNA Isolation Mini Kit

2 User Manual TM FavorPrep Soil RNA Isolation Mini Kit Cat.No. : FASRK 000, 4 preps FASRK 001, 50 Preps (For Research Use Only) Kit Contents: FASRK 000 FASRK 001 (4 preps_sample) (50 preps) Glass Beads 2 g 24 g SR1 Buffer 5 ml 60 ml SR2 Buffer 0.3 ml 4 ml SR3 Buffer 0.6 ml 8 ml SR4 Buffer 1.5 ml 20 ml Wash Buffer (concentrate) * 1.5 ml 15 ml RNase-free water 1.5 ml 20 ml RNA Mini Column 4 pcs 50 pcs Collection Tube 8 pcs 100 pcs Elution Tube (1.5 ml tube) 4 pcs 50 pcs 2.0 ml tube 4 pcs 50 pcs User Manual 1 1 * Preparation of Wash Buffer by adding ethanol (96% ~100%) for first use: Cat. No: FASRK 000 FASRK 001 ethanol volume for Wash Buffer 6 ml 60 ml Specification: Principle: spin column (silica membrane) Sample: 0.25 ~ 1 g Operation time: < 60 min Elution volume: 40 µl Brief Procedure: soil sample Glass beads SR1 Buffer SR2 Buffer Important Notes: 1. Make sure everything is RNase-free when handling RNA. 2. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers. 3. Add indicated volume of ethanol (96-100%) to Wash Buffer before use. 4. Phenol and chloroform are hazardous to human health. perform the procedures involving phenol or chloroform in a chemical fume hood. 5. Preheat RNase free water to 60 C for elution step. centrifuge Water-saturated phenol Chloroform plus-vortexing for 5 minutes at 4,000 x g for 10 minutes RNA dissolve Additional material to be provided by user: Ethanol (RNase-free, 96~100%) Isopropanol Water-saturated phenol Chloroform Microcentrifuge for 1.5 ml and 2.0 ml tube capable of at least 13,000 x g fugr for 15 ml tube capable of at least 4,000 X g Water Bath or Dry Bath 1.5 ml centrifuge tube 15 ml centrifuge tube SR3, room Temp., 2 min SR4, Ethanol (96~100%) Binding Wash X2 RNA Elution 1 v 0815

3 General Protocol: Please Read Important Notes Before Starting Following Steps. 1. Add 400 mg of Glass Beads (provided) and 1 g of soil sample to a 15 ml centrifuge tube (not provided). --If the sample is liquid, add 500 µl of sample to the 15 ml Beads Tube. 2. Add 1 ml of SR1 Buffer to the sample, vortex at maximum speed for 1 minute. 3. Add 50 µl of SR2 Buffer to the sample, vortex at maximum speed for 1 minute. 4. Add 1 ml of water saturated-phenol and 200 µl of chloroform to the sample, mix well by plus-vortexing for 5 minutes to lysis the sample. Caution! Phenol and chloroform are hazardous, performing Step 4 and Step 5 in a chemical fume hood. 5. at 4,000 x g for 10 minutes to form aqueous and organic phase. 6. Carefully transfer the upper aqueous phase to a 2.0 ml centrifuge tube (provied). Measure the volume of the upper aqueous. -- Carefully transfer the upper aqueous phase and do not disrupt the inter phase that consist of phenol and chlorofrom. -- Avoid pipetting any debris and pellet. 7. Add 1 volume of isopropanol, vortex to mix well. centrifuge at full speed (13,000 x g) for 10 min to form a pellet. 8. Carefully discard the supernatant and invert the tube on the paper towel for 5 min to remove residual liquid. -- Do not disrupt the pellet when discard the supernatant. -- Depending on the soil type, the dark color of pellet is not consist. 9. Add 200 µl of RNase-free ddh2o, vortex to dissolve the pellet completely. -- Incubate the tube at 45 C for 10 min if the pellet is hard to dissolve. 10. Add 100 µl of SR3 Buffer to the sample, mix well by vortexing. Incubate the sample at room temperature for 2 minutes. -- Note: SR3 Buffer must be suspended completely by vigorously vrotexing before every using. -- use 1ml pipettor and cut off the end of 1 ml tip to make it easier for pipetting the SR3 Buffer. 11. at full speed (13,000 x g) for 2 minutes. 12. Carefully transfer the clarified lysate to a 1.5 ml microcentrifuge (not provied). --Avoid pipetting any debris and pellet. 13. Add 250 µl of SR4 Buffer and 250 µl of ethanol (RNase-free, 96~100%) to the clarified lysate, mix thoroughly by pulse-vortexing. 14. Place a RNA Mini Column into a Collection Tube and transfer all of the sample mixture to the RNA Column. at full speed (13,000 x g) for 1 min then discard the flow-through. Place the RNA Column to a new Collection Tube. 15. Add 650 µl of Wash Buffer (ethanol added) to RNA Mini Column. at full speed (13,000 x g) for 1 min then discard the flow-through. --Make sure that ethanol (RNase-free, 96~100%) has been added into Wash Buffer when first open. 16. Repeat step 15 for one more time. 17. at full speed (13,000 x g) for an additional 3 min to dry the RNA Mini Column. --Important step! This step will avoid the residual liquid to inhibit subsequent enzymatic reactions. 18. Place RNA Column into a Elution Tube, Add 40 µl of pre-heated RNase-free ddh2o to the membrane center of the RNA Mini Column. Stand the RNA Column for 2 min at room temperature. --Important step! For effective elution, make sure that the RNase-free ddh2o is dispensed onto the membrane center and is absorbed completely. 19. at full speed (13,000 x g) for 1 min to elute RNA. 2

4 FavorPrep Soil DNA Isolation Mini Kit

5 User Manual Kit Contents: TM FavorPrep Soil DNA Isolation Mini Kit FASOI 000 (4 preps_sample) FASOI 001 (50 preps) FASOI (100 preps) Glass Beads 1 g 12 g 25 g SDE1 Buffer 3.6 ml 40 ml 70 ml SDE2 Buffer 1.2 ml 15 ml 25 ml SDE3 Buffer 1.2 ml 15 ml 30 ml SDE4 Buffer 1.5 ml 25 ml 40 ml Wash Buffer (concentrate) * 1.5 ml 20 ml 40 ml Elution Buffer 1.5 ml 25ml 50 ml SDE Mini Column 4 pcs 50 pcs 100 pcs Collection Tube 8 pcs 100 pcs 200 pcs Elution Tube 4 pcs 50 pcs 100 pcs Bead tube 4 pcs 50 pcs 100 pcs User Manual Cat.No. : FASOI 000, 4 preps FASOI 001, 50 Preps FASOI 001-1, 100 Preps (For Research Use Only) * Preparation of Wash Buffer for first use: ethanol volume for Wash Buffer Cat. No: FASOI 000 FASOI 001 FASOI ml 80 ml 160 ml Specification: Principle: spin column (silica membrane) Sample: 0.25 ~ 0.5 g Operation time: < 60 min Elution volume: 50 ~ 200 µl Brief Procedure: soil sample Lysis: SDE1, 70 C, 10 min Important Notes: 1. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers. 2. Check SDE1 Buffer before use, Warm SDE1 Buffer at 60 C for 10 minutes if any precipitate formd. 3. Add indicated volume of ethanol (96-100%) to Wash Buffer before use. 4. Prepare a heating block or a water bath to 70 C. If DNA is isolated from gram positive bacteria, prepare a heating block or a water bath to 95 C for another incubation. 5. All centrifuge steps are done at full speed (~18,000 x g) in a microcentrifuge. 6. Preheat Elution Buffer or ddh2o to 60 C for elution step. SDE2, on ice, 5 min Add Isopropanol DNA dissolve SDE3, room Temp., 2 min SDE4, Ethanol (96~100%) General Protocol: Please Read Important Notes Before Starting Following Steps. Binding 1. Add 200 mg of Glass Beads into a 2.0 ml Bead Tube (provided). Transfer 0.25 ~ 0.5 g of soil sample into Bead Tube then place on ice. --If the sample is liquid, add 200 µl of sample into a 2.0 ml Beads Tube. Wash X2 2. Add 600 µl of SDE1 Buffer to the sample, vortex at maximum speed for 5 minutes. Incubate the sample at 70 C for 10 minutes and vortex the Elution sample twice during the incubation. --For isolation of DNA from gram positive baceria, do a further incubation at 95 C for 5 minutes. 3. Briefly spin the tube to remove drops from the inside of the lid. 4. Cool down the sample mixture and add 200 µl of SDE2 Buffer. Mix well by vortexing. Incubate the sample on ice for 5 minutes. 5. at full speed (~ 18,000 x g) for 5 minutes. 6. Carefully transfer the clarified supernatant to a 1.5 ml microcentrifuge tube (not provied). Measure the volume of the supernatant. --Avoid pipetting any debris and pellet. 7. Add 1 volume of isopropanol and vortex to mix well. centrifuge at full speed for 10 min to pellet DNA. -- For example: If the clarified lysate volume is 450 µl, add 450 µl of isopropanol to the clarified lysate. 8. Carefully discard the supernatant and invert the tube on the paper towel for 1 min to remove residual liquid. --Do not disrupt the pelle. 9. Add 200 µl of pre-heated Elution Buffer or ddh2o, vortex to dissolve the DNA pellet completely. 10. Add 100 µl of SDE3 Buffer to the sample, mix well by vortexing. Incubate the sample at room temperature for 3 minutes. --Note: SDE3 Buffer must be suspended completely by vigorously vrotexing before every using. - - Cut off the end of 1 ml tip to make it easier for pipetting the SDE3 Buffer. 1 v 0515

6 11. at full speed for 2 minutes. 12. Carefully transfer the supernatant to a 1.5 ml microcentrifuge (not provied). And measure the volume of the supernatant. --Avoid pipetting any debris and pellet. 13. (Optional) If RNA-free DNA is required, add 1 µl of 100 mg/ml RNase A (not provided) to the sample and mix well. Incubate at room temperature for 2 min. 14. Briefly spin the tube to remove drops from the inside of the lid. 15. Add 1 volume of SDE4 Buffer and 1 volume of ethanol (96~100%). Mix thoroughly by pulse-vortexing. For example: If the clarified lysate volume is 250 µl, add 250 µl of SDE4 Buffer and 250 µl of ethanol (96~100%) to the sample. 16. Place a SDE Column into a Collection Tube and transfer all of the sample mixture to the SDE Column. at full speed for 1 min then discard the flow-through then place the SDE Column into a new Collection Tube. 17. Add 750 µl of Wash Buffer (ethanol added) to the SDE Column. at full speed for 1 min then discard the flow-through. --Make sure that ethanol (96~100%) has been added into Wash Buffer when first use. 18. Repeat step at full speed for an additional 3 min to dry the SDE column. --Important step! This step will avoid the residual liquid to inhibi subsequent enzymatic reactions. 20. Place the SDE Column into a 1.5 ml microcentrifuge tube (not provided). Add 50 ~ 200 µl of preheated Elution Buffer or ddh2o to the membrane center of the SDE Column. Stand the SDE Column for 2 min at room temperature. --Important step! For effective elution, make sure that the Elution Buffer or ddh2o is dispensed onto the membrane center and is absorbed completely. 21. at full speed for 1 min to elute DNA. Troubleshooting Problem Possible reasons Soutions Low or no yield of genomic DNA Sample stored incorrectly Low amount of cells in the sample Poor cell lysis Poor cell lysis because of insufficient beads beating time Insufficient binding of DNA to column s membrane Ethanol is not added into sample lysate before DNA binding Ethanol and sample lysate did not mix well before DNA binding Poor quality of genomic DNA Store the stool sample at -20 C. Increase the sample size Extend the beads beating time. Make sure that the correct volumes of ethanol ( %) is added into the sample lysate before DNA binding. Make sure that Ethanol and sample lysate have been mixed completely before DNA binding Incorrect preparation of Wash Buffer W1/W2 Ethanol is not added into Wash Buffer when first use The volume or the percentage of ethanol is not correct for adding into Wash Buffer Elution of DNA is not efficient ph of water (ddh2o) for elution is acidic Make sure the ph of ddh2o is between Use Elution Buffer (provided) for elution. Elution Buffer or ddh2o is not completely absorbed by column membrane Make sure that the correct volumes of ethanol ( %) is added into Wash Buffer when first use. Make sure that the correct volumes of ethanol ( %) is added into Wash Buffer when first use. After Elution Buffer or ddh2o is added, stand the SD Column for 5 min before centrifugation. A260/A280 ratio of eluted DNA is low A260/A280 ratio of eluted DNA is high Poor cell lysis Poor cell lysis because of insufficient beads beating time A lot of residual RNA in eluted DNA Extend the beads beating time. Add 8 µl of RNase A (50 mg/ml) to the eluate and incubate at 37 C for 10 minutes. After incubation, add 200 µl of SD2 Buffer and 200 µl of ethanol (96~100%), mix well by plus -vortexing. Then follow the general Protocol starting from step 7. 2

7 FavorPrep Soil DNA Isolation Midi KitKit

8 TM FavorPrep Soil DNA Isolation Midi Kit User Manual Cat. No.: FASOI 002 (24 Preps) For Research Use Only v.1107

9 Introduction The FavorPrep Soil DNA Isolation Midi Kit is designed for isolation of total DNA from 1 ~10 g of soil sample. The inhibitors of the downstream PCR or enzymatic reactions will be removed with the sequent buffers system of this kit. The entire procedure is not required the phenol-chloroform extraction and can be finished within 90 min. The purified DNA is ready for PCR and other downstream application. Kit Contents 18. at 2,500 ~ 4,000 x g at room temperature for an additional 10 min to dry the SDE Midi column. --It might be necessary to dry the column futher by placing the column in a vacuum oven at 70 C for 10 minutes. --Important step! This step will avoid the residual liquid to inhibit subsequent enzymatic reactions. 19. Place SDE Midi Column into a new 50 centrifuge tube, Add 1~ 2 ml of preheated Elution Buffer or ddh2o to the membrane center of the SDE Midi Column. Stand the SDE Midi Column for 5 min at room temperature. --Important step! For effective elution, make sure that the Elution Buffer or ddh2o is dispensed onto the membrane center and is absorbed completely. 20. at 2,500 x g at room temperature for 2 min to elute DNA. FASOI002 (24 Preps) Glass Beads SDE1 Buffer SDE2 Buffer SDE3 Buffer SDE4 Buffer Wash Buffer (concentrated) Elution Buffer SDE Midi Column 55 g 200 ml X2 135 ml 30 ml 85 ml 40 ml* X2 120 ml 24 pcs Elution Tube (50 ml centrifuge tube) 24 pcs User Manual 1 *Add 160 ml ethanol (96~100%) to Wash Buffer when first open. 1 6

10 10. Add 1 ml of SDE3 Buffer to the sample, mix well by vortexing. Incubate the sample at room temperature for 3 minutes. --Note: SDE3 Buffer must be suspended completely by vigorously vrotexing before every using. --Use 1ml pipettor and cut off the end of 1 ml tip to make it easier for pipetting the SDE3 Buffer. 11. at 2,500 x g at room temperature for 5 minutes. 12. Carefully transfer the clarified lysate to a 15 ml or 50 ml microcentrifuge (not provied). And measure the volume of the clarified lysate. --Avoid pipetting any debris and pellet. 13. (Optional) If RNA-free DNA is required, add 4 µl of 100 mg/ml RNase A (not provided) to the sample and mix well. Incubate at room temperature for 2 min. 14. Add 1 volume of SDE4 Buffer and 1 volume of ethanol (96~100%) to the clarified lysate, mix thoroughly by pulse-vortexing. For example: If the clarified lysate volume is 2.5 ml, add 2.5 ml of SDE4 Buffer and 2.5 ml of ethanol (96~100%) to the clarified ltsate. 15. Place the SDE Midi Column in a 50 ml centrifuge (Ex: Falcon 50 ml) and transfer all of the sample mixture to the SDE Midi Column. 16. at 2,500 x g at room temperature for 2 min then discard the flow-through. Then place the SDE Midi Column back in 50 ml centrifuge tube. 17. Add 7 ml of Wash Buffer (ethanol added) to SDE Midi Column. at 2,500 x g at room temperature for 2 min then discard the flow-through. And repeat this step for one more time. --Make sure that ethanol (96~100%) has been added into Wash Buffer when first open. Applications: PCR, Real-Time PCR Infection disease research Sample amount: Sample: up to 10 g of soil sample Handing time: 90 minutes Elution Volume: 2 ml Important Notes 1. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers. 2. Check SDE1 Buffer before use, Warm SDE1 Buffer at 60 C for 10 minutes if any precipitate formd. 3. Add 160 ml of ethanol (96-100%) to Wash Buffer when first open. 4. Prepare a water baths to 70 C before the operation. 5. All centrifuge steps are done at full speed (14,000 rpm or 10,000 x g) in a microcentrifuge. 6. Preheat Elution Buffer or ddh2o to 60 C for elution step. 5 2

11 Brief Procedure General Protocol: soil sample Please Read Important Notes Before Starting Following Steps. Lysis: SDE1, 70 C, 10 min SDE2, on ice, 5 min DNA Precipitation (Isopropanol) DNA dissolve SDE3, room Temp., 2 min SDE4, Ethanol (96~100%) Binding Wash X2 1. Transfer up to 10 g of soil sample to a 50 ml centrifuge tube. (not provided) 2. Weigh 2 g of Glass bead and mix with soil sample. 3. Add 15 ml of SDE1 Buffer to the sample, vortex at maximum speed for 5 minutes. Incubate the sample at 70 C for 10 minutes and vortex the sample twice during the incubation. --For isolation of DNA from gram positive baceria, do a further incubation at 95 C for 5 minutes. 4. Cool down the sample and add 5 ml of SDE2 Buffer to the sample, mix well by vortexing. Incubate the sample on ice for 5 minutes. 5. at 2,500 x g for 5 minutes at room temperature. 6. Carefully transfer the clarified lysate to a 50 ml centrifuge tube (not provied). And measure the volume of the clarified lysate. --Avoid pipetting any debris and pellet. 7. Add 1 volume of isopropanol, vortex to mix well. centrifuge at 15,000 x g at 4 C for 30 min to precipate DNA. -- For example: If the clarified lysate volume is 12 ml, add 12 ml of isopropanol to the clarified ltsate. 8. Carefully discard the supernatant and invert the tube on the paper towel for 5 min to remove residual liquid. --Do not disturb the pellet. 9. Add 2 ml of pre-heated Elution Buffer or ddh2o, vortex to dissolve the pellet completely. Elution 3 4