96-well Genomic DNA Extraction Kit. Protocol Book. (Deep Well) Ver RBP96B-2

Transcription

1 96-well Genomic DNA Extraction Kit (Deep Well) Protocol Book RBP96B-2 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 96 well Genomic DNA Extraction Kit (deep Well) Cat.No. RBP96B-2 Blood Protocol (Vaccum Protocol) Tissue Protocol (Vaccum Protocol) For Blood or Body Fluids (Spin Protocol) For Tissue (Spin Protocol)

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6 96 well Genomic DNA Extraction Kit (Deep Well) Cat.No. RBP96B-2 Kit Contents Centrifuge requires two plates for balance at all times Cat.No. RBP96B-2 2 Plates GT Buffer...60ml GB Buffer...50ml W1 Buffer...50ml x2 Wash Buffer (concentrated)*...40ml Elution Buffer...60ml Proteinase K**...40mg 96-Well DNA Binding Plate...2pcs 96-Well 2ml Sample Plate...4pcs 96-Well PCR Plate...2pcs Adhesive film...10pcs Sample : 200 μl of whole blood 25mg of animal tissue cultured cells 10 9 bacterial culyures Yields: Up to 50 μg of genomic DNA Operation Time: < 60 mins Elution Volume: μl * Add 160 ml of ethanol (96-100%) to Wash Buffer prior to the initial use. ** Add 4.0 ml ddh2o to the Proteinase K bottle and mix vortexing. *** Store prepared Proteinase K (10mg/ml) at 4 C 1 96 well Genomic DNA Extraction Kit (Deep Well)

7 Description 96-Well Genomic DNA Extraction Kit is designed for high-throughput purification of total DNA (including genomic, mitochondrial and viral DNA) from whole blood and a variety of animal tissues or cells. The method use proteinase K and a chaotropic salt, guanidine hydrochloride to lyse cells and degrade protein, then DNA in chaotropic salt is bonded to glass fiber matrix of plate. After washing off the contaminants, the purified DNA is eluted by low salt elution buffer or water. The entire procedure can be completed in one hour without phenol/ chloroform extraction and alcohol precipitation. The kits can be used for manual filtration or with robotic handing systems and purified DNA with approximately kb is suitable for PCR or other enzymatic reactions. Quality Control The quality of 96-Well Genomic DNA Kit is tested on a lot-to-lot basis. The purified DNA is checked by agarose gel analysis and quantified with spectrophotometer. Caution GB Buffer and W1 Buffer contain guanidine hydrochloride, which is harmful, and irritant agent. During operation, always wear a lab coat, disposable gloves, and protective goggles. 96 well Genomic DNA Extraction Kit (Deep Well) 2

8 Blood Protocol (Vacuum Protocol) Cell lysis 1. Add 200 μl GB Buffer and 20 μl Proteinase K (10 mg/ml) to each well of a 96-Well 2 ml plate. 2. Apply 200 μl of blood sample to each well and mix by shaking. Seal with Adhesive Film. 3. Incubate at 70 C for 20 minutes. 4. Preheat required Elution Buffer (100 μl per sample) at 70 C. (For DNA elution) DNA Binding 5. Add 200μl of ethanol to each well of sample lysate from precious step. Mix immediately by pipetting 5-10 times. 6. Place a 96-Well DNA Binding Plate on top of the vacuum manifold. 7. Transfer the lysate mixture to 96-Well DNA Binding Plate. 8. Apply vacuum at 10 inches Hg for 30 seconds until wells have emptied. Wash 9. Add 250 μl of W1 Buffer to each well of the 96-Well DNA Binding Plate. 10. Apply vacuum at 10 inches Hg for 30 seconds until wells have emptied. 11. Add 250 μl of Wash Buffer (ethanol added) to each well of the 96-well DNA Binding Plate to wash again. 12. Apply vacuum at 10 inches Hg for 30 seconds until wells have emptied. 13. Apply vacuum for additional 10 min (or incubate at 60 C) to remove ethanol residue well Genomic DNA Extraction Kit (Vacuum Protocol)

9 DNA Elution Standard elution volume is 100μl. If less sample is to be used, reduce the elution volume (50-70 μl) to increase DNA concentration. 14. Transfer the DNA Binding Plate on a clean 96-Well PCR plate. 15. Add 70μl of preheated Elution Buffer in the center of each well of DNA Binding Plate. 16. Stand for 3 minutes until Elution Buffer or water absorbed by the matrix. 17. Place the combined plates in a rotor bucket and centrifuge at 3,500 rpm for 5 min to elute purified DNA into the 96-Well PCR plate. 18. Seal with Adhesive Film and store 4 C or -20 C. 96 well Genomic DNA Extraction Kit (Vacuum Protocol) 4

10 Tissue Protocol (Vacuum Protocol) Cell lysis 1. Add 200 μl GT Buffer and 20 μl Proteinase K (10 mg/ ml) to each well of a 96-Well 2 ml sample plate (not provided). 2. Cut up to 25 mg of animal tissues (or 0.5 cm of mouse tail) and transfer into each well of 96-Well 2 ml sample plate. Seal with Adhesive Film. 3. Incubate the plate with shaking at 60 C for 1-2 hours to lyse the sample. 4. If RNA-free genomic DNA is required, add 5 μl of RNase A (50 mg/ml, not provided) to each well and incubate at room temperature for 4 minutes. 5. Add 200 μl GB Buffer to each well and mix by shaking (seal with Adhesive Film.). 6. Incubate at 70 C for 20 minutes until the sample lysate is clear. 7. Preheat required Elution Buffer (50 μl per sample) at 70 C. (For DNA elution) 8. If there are insoluble material present following incubation, centrifuge the plate for 5 minutes at full speed and transfer the supernatants to a new 96-Well 2 ml sample plate (not provided). DNA Binding 9. Add 200μl of ethanol to each well of sample lysate from precious step. Mix immediately by pipetting 5-10 times. 10. Place a 96-Well DNA Binding Plate on top of the vacuum manifold. 11. Transfer the lysate mixture to 96-Well DNA Binding Plate. 12. Apply vacuum at 10 inches Hg for 30 seconds until wells have emptied well Genomic DNA Extraction Kit (Vacuum Protocol)

11 Wash 13. Add 250 μl of W1 Buffer to each well of the 96-Well DNA Binding Plate. 14. Apply vacuum at 10 inches Hg for 30 seconds until wells have emptied. 15. Add250μl of Wash Buffer (ethanol added) to each well of the 96-well DNA Binding Plate to wash again. 16. Apply vacuum at 10 inches Hg for 30 seconds until wells have emptied. 17. Apply vacuum for additional 10 min (or incubate at 60 C for 10minutes) to remove ethanol residue. DNA Elution Standard elution volume is 100μl. If less sample is to be used, reduce the elution volume (50-70 μl) to increase DNA concentration. 18. Transfer the DNA Binding Plate on a clean 96-Well PCR plate. 19. Add 70 μl of preheated Elution Buffer in the center of each well of DNA Binding Plate. 20. Stand for 3 minutes until Elution Buffer or water absorbed by the matrix. 21. Place the combined plates in a rotor bucket and centrifuge at 3,500 rpm for 5 min to elute purified DNA into the 96-Well PCR plate. Seal with Adhesive Film and store 4 C or -20 C. 96 well Genomic DNA Extraction Kit (Vacuum Protocol) 6

12 For Blood or Body Fluids (Spin Protocol) Cell lysis 1. Add 200 μl GB Buffer and 20 μl Proteinase K (10 mg/ml) to each well of a 96-Well 2 ml plate. 2. Apply 200 μl of blood sample to each well and mix by shaking. Seal with Adhesive Film. 3. Incubate at 70 C for 20 minutes. 4. Preheat required Elution Buffer (100 μl per sample) at 70 C. (For DNA elution) DNA Binding 5. Add 200μl of ethanol to each well of sample lysate from precious step. Mix immediately by pipetting 5-10 times. 6. Place a 96-Well DNA Binding Plate on top of a new 96-well 2 ml Sample Plate. 7. Apply 250~300μl lysate mixture to 96-Well DNA Binding Plate. 8. Place in a rotor bucket and centrifuge for 5 min at 3,500 rpm. 9. Discard the flow-through and return the 96-Well DNA Binding Plate to the 96-well 2ml Sample Plate. 10. Repeat step 7~step 9 to bind the sample again well Genomic DNA Extraction Kit (Spin Protocol)

13 Wash 11. Add 250 μl of W1 Buffer to each well of the 96-Well DNA Binding Plate. 12. Centrifuge 5 min at 3,500 rpm in a centrifuge. 13. Discard the flow-through and return the 96-Well DNA Binding Plate to the 96-well 2ml Sample Plate. 14. Add 250 μl of Wash Buffer (ethanol added) to each well of the 96-well DNA Binding Plate. 15. Centrifuge 5 min at 3,500 rpm in a centrifuge. 16. Discard the flow-through and return the 96-Well DNA Binding Plate to the 96-well 2ml Sample Plate. 17. Repeat step 14~step 16 to wash the sample again. 18. Centrifuge 10 min at 3,500 rpm in a centrifuge to remove ethanol residue. DNA Elution Standard elution volume is 100μl. If less sample is to be used, reduce the elution volume (50-70 μl) to increase DNA concentration. 19. Place a clean 96-well PCR Plate on top of a clean 96-well 2 ml Sample Plate for support. Transfer the 96-Well DNA Binding Plate on to the top of the 96-well PCR Plate. 96-well PCR Plates are ideal for handling storage of small elution volumes. 20. Add 100 μl of Elution Buffer or ddh 2 O (ph ) into the center of the membrane. 21. Allow to stand for 2 minutes until Elution Buffer or water has been absorbed by the matrix. Place the entire assembly into the rotor bucket. 22. Centrifuge for 5 minutes at 3,500 rpm in a centrifuge to elute purified DNA into the 96-well PCR Plate. Seal with Sealing Film and store at 4 C or -20 C 96 well Genomic DNA Extraction Kit (Spin Protocol) 8

14 For Tissue (Spin Protocol) Cell lysis 1. Add 200 μl GT buffer and 20 μl Proteinase K (10 mg/ml) to each well of a 96-Well 2ml Sample Plate. 2. Cut up to 25 mg of animal tissues (or 0.5 cm of mouse tail) and transfer into each well of 96-Well 2 ml Sample Plate. Seal with Adhesive Film. 3. Incubate the plate with shaking at 60 C for 1-2 hours to lyse the sample. 4. If RNA-free genomic DNA is required, add 5 μl of RNase A (50 mg/ml, not provided) to each well and incubate at room temperature for 4 minutes. 5. Add 200 μl GB Buffer to each well and mix by shaking (seal with Adhesive Film.) 6. Incubate at 70 C for 20 minutes until the sample lysate is clear. 7. Preheat required Elution Buffer (100 μl per sample) at 70 C. (For DNA elution) 8. If there are insoluble material present following incubation, centrifuge the plate for 5 minutes at full speed and transfer the supernatants to a new 96-Well 2 ml Sample Plate (not provided). DNA Binding 9. Add 200μl of ethanol to each well of sample lysate from precious step. Mix immediately by pipetting 5-10 times. 10. Place a 96-Well DNA Binding Plate on top of a new 96-well 2 ml Sample Plate. 11. Apply 250~300μl lysate mixture to 96-Well DNA Binding Plate. 12. Place in a rotor bucket and centrifuge for 5 min at 3,500 rpm. 13. Discard the flow-through and return the 96-Well DNA Binding Plate to the 96-well 2ml Sample Plate. 14. Repeat step 11~ 13 to bind the sample again well Genomic DNA Extraction Kit (Spin Protocol)

15 Wash 15. Add 250 μl of W1 Buffer to each well of the 96-Well DNA Binding Plate. 16. Centrifuge 5 min at 3,500 rpm in a centrifuge. 17. Discard the flow-through and return the 96-Well DNA Binding Plate to the 96-well 2ml Sample Plate. 18. Add 250 μl of Wash Buffer (ethanol added) to each well of the 96-well DNA Binding Plate. 19. Centrifuge 5 min at 3,500 rpm in a centrifuge. 20. Discard the flow-through and return the 96-Well DNA Binding Plate to the 96-well 2ml Sample Plate. 21. Repeat step 18~ 20 to wash the sample again. 22. Centrifuge 10 min at 3,500 rpm in a centrifuge to remove ethanol residue. DNA Elution Standard elution volume is 100μl. If less sample is to be used, reduce the elution volume (50-70 μl) to increase DNA concentration. 23. Place a clean 96-well PCR Plate on top of a clean 96-well 2 ml Sample Plate for support. Transfer the 96-Well DNA Binding Plate on to the top of the 96-well PCR Plate. 96-well PCR Plates are ideal for handling storage of small elution volumes. 24. Add 100 μl of Elution Buffer or ddh2o (ph ) into the center of the membrane. 25. Allow to stand for 2 minutes until Elution Buffer or water has been absorbed by the matrix. Place the entire assembly into the rotor bucket. 26. Centrifuge for 5 minutes at 3,500 rpm in a centrifuge to elute purified DNA into the 96-well PCR Plate. Seal with Sealing Film and store at 4 C or -20 C. 96 well Genomic DNA Extraction Kit (Spin Protocol) 10

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