Mag-Bind Blood DNA KF 96 Kit. M x 96 preps M x 96 preps

Transcription

1 Mag-Bind Blood DNA KF 96 Kit M x 96 preps M x 96 preps June 2013

2 Mag-Bind Blood DNA KF 96 Kit Table of Contents Introduction and Principle...2 Kit Contents and Storage...3 Preparing Reagents...4 KingFisher Pipette Instructions...5 Recommended Settings...6 Mag-Bind Blood Protocol...7 Mag-Bind Blood with Incubator Protocol...9 Mag-Bind Blood with KingFisher ml Protocol...10 Determination of Yield and Quality...12 Troubleshooting Guide...13 Manual Revision: June 2013 Innovations in nucleic acid isolation 1

3 Introduction and Principle Introduction Mag-Bind Blood DNA KF 96 Kit allows rapid and reliable isolation of high-quality genomic DNA from μl blood samples. The system combines the reversible nucleic acidbinding properties of Mag-Bind magnetic particles with the time-proven efficiency of Omega Bio-tek s blood DNA isolation system to provide a fast and convenient blood DNA isolation method. The magnetic particles technology provides high-quality DNA that is suitable for direct use in most downstream applications, such as amplification and enzymatic reactions. Principle Mag-Bind DNA Isolation Kits use the reversible binding properties of the Mag-Bind paramagnetic particles to provide a fast and flexible method for isolating genomic DNA from different whole Blood and cultured cells. Samples are first lysed with a specially formulated buffer containing detergent in the presence of Proteinase K. After adjust the binding condition, the sample was mixed with Mag-Bind particles and the genomic DNA was bound to the surface of Mag-Bind magnetic particles. Proteins, polysaccharides, and cellular debris are efficiently washed away with few wash steps. Pure DNA is then eluted in water or low ionic strength buffer. Purified DNA can be directly used in downstream applications without the need for further purification. New in this edition: Proteinase K is now supplied in a liquid form eliminating the step to respuspend prior to use. Proteinase K Solution can also be stored at room temperature for 12 months. Proteinase Storage Buffer is no longer included in the kit. 2

4 Kit Contents Product M M Purification 1 x 96 4 x 96 Mag-Bind Particles CND 1.1 ml 4.4 ml Binding Enhancer 1.1 ml 4.4 ml MSL Buffer 35 ml 140 ml SPM Wash Buffer 45 ml 144 ml QMP Buffer 30 ml 120 ml Elution Buffer 15 ml 60 ml Proteinase K Solution 3 ml 12 ml User Manual 1 1 Warning: MSL Buffer and QMP Buffer contains a chaotropic salt. Please wear gloves, and appropriate eyewear while performing this procedure. Storage and Stability All of the Mag-Bind Blood DNA KF 96 Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows. Mag-Bind Particles CND and Binding Enhancer should be stored at 2-8 C for long-term use. Proteinase K Solution can be stored at room temperature for up to 12 months. For long-term storage, store Proteinase K Solution at 2-8 C. All remaining components should be stored at room temperature. During shipment or storage in cool ambient conditions, precipitates may form in MSL Buffer. Dissolve such deposits by warming the solution at 37 C and gently shaking. 3

5 Preparing Reagents Dilute SPM Wash Buffer with 100% ethanol as follows as store at room temperature. Product Ethanol to be Added M ml M ml Dilute QMP Buffer with isopropanol as follows and store at room temperature. This mixture can only be stored for two weeks. Aliquot if necessary. Product Isopropanol to be Added M ml M ml 4

6 KingFisher Pipetting Instructions Pipetting Instructions for KingFisher 96 or KingFisher Flex with the Mag-Bind Blood DNA protocols. Plate Type * Plate Content Reagent Volume A 1 Sample/ Lysate according to protocols below. A 2 QMP Buffer 500 µl A 3 SPM Wash Buffer 600 µl A 4 SPM Wash Buffer 600 µl B 5 Elution Buffer 100 µl A 6 Tip Sleeve * A= KingFisher 96 DW Plate, B=KingFisher 96 KF Plate Prepare lysate by following the use instruction based on sample type. Add 500 µl QMP Buffer to Plate 2. Add 600 µl SPM Wash Buffer to Plate 3. Add 600 µl SPM Wash Buffer to Plate 4. Add 100 µl Elution Buffer to Plate 5. 5

7 Recommended Settings The following recommendation are based upon Thermo Fisher s KingFisher software version Contact the instrument s manufacturer for updated software if all speeds are not available. This protocol is designed to show the recommended settings for all binding, washing, drying and elution steps in a 96-well format, lysis steps are not included in this protocol. Step 1: Binding Beginning of Step: No Action Binding Parameters: 05:00 at Superfast; Collect Beads at Count of 8 Step 2: High Salt Wash Step Beginning of Step: Release Beads, 00:10 at Fast, Wash Time: 01:00, Very Fast; Collect Beads at Count of 8 Step 3: Ethanol Wash Step Beginning of Step: Release Beads, 00:10 at Fast, Wash Time: 1:00, Half Mix Collect Beads at Count of 8 Step 4: Ethanol Wash Step Beginning of Step: Release Beads, 00:10 at Fast, Wash Time: 1:00, Half Mix Collect Beads at Count of 8 Step 5: Dry Step 05:00 Outside Wells/tubes Step 6: Elution Beginning of Step: Release Beads 00:10 at Fast Elution Time: 00:00 at Fast Heating Parameters: Preheat, 08:00 at 70 C Heating Action: Mix at Fast Post Mix: 02:00 at Grind Mix, Collect Beads at Count of 8 Disposal Plate: Ethanol Wash 1 6

8 Mag-Bind Blood DNA Protocol Mag-Bind Blood DNA Protocol (1-200 µl Blood) This protocol is for the isolation of whole Blood on the KingFisher Flex 96 or KingFisher 96 instrument. This protocol requires the incubation of the blood to be done prior to adding to the KingFisher instrument. Please see following protocol for the incubation (Step 4) to be done on the KingFisher flex instrument Materials Supplied by User: KingFisher Flex 96 or KingFisher 96 Incubator capable of 70 C Vortexer Multi-channel pipettor KingFisher deep-well plates KingFisher 96 plate KingFisher deep-well tip comb Sealing film 100% Ethanol Isopropanol Optional: 10 mm Tris-HCl or PBS Optional: RNase A Before Starting: Set incubator to 70 C Prepare SPM Wash Buffer and QMP Buffer according to the Preparing Reagents section on Page 4 1. Add blood sample to a KingFisher deep-well plate. Bring the volume up to 200 μl with 10 mm Tris-HCl, PBS, or Elution Buffer provided with this kit. 2. Add 20 µl Proteinase K Solution and 5 µl RNase A (not provided) to the samples. 3. Add 220 µl MSL Buffer and 5 µl Binding Enhancer to the samples and seal the plate with sealing film (not provided). Vortex for 20 seconds. Note: MSL Buffer and Binding Enhancer can be made as a master mix. For 96 samples add 22 ml MSL Buffer and 5 ml Binding Enhancer. Add 225 µl to each sample. 7

9 Mag-Bind Blood DNA Protocol 4. Incubate at 70 C for 10 minutes. 5. Add buffers to appropriate plates according to the KingFisher Pipetting Instructions (Page 5). 6. Remove the sealing film. Add 305 μl ethanol and 10 μl Mag-Bind Particles CND to each sample. 7. Press start on KingFisher 96 whole blood protocol and load plates accordingly. 8. Remove plate containing genomic DNA and store at - 20 C. 8

10 KF with Included Incubator Protocol Mag-Bind Blood DNA (1-200 µl Blood) with Incubation on KingFisher 96 This protocol is for the isolation of whole blood on the KingFisher Flex 96 or KingFisher 96 instrument. This protocol requires the incubation of the blood to be done on the KingFisher instrument. Materials Supplied by User: KingFisher Flex 96 or KingFisher 96 Incubator capable of 70 C Vortexer Multi-channel pipettor KingFisher deep-well plates KingFisher 96 plate KingFisher deep-well tip comb Sealing film 100% Ethanol Isopropanol Optional: 10 mm Tris-HCl or PBS Optional: RNase A Before Starting: Prepare SPM Wash Buffer and QMP Buffer according to the Preparing Reagents section on Page 4 Add buffers to appropriate plates according to the KingFisher Pipetting Instructions on Page 5 1. Add blood sample to a KingFisher deep-well plate. Bring the volume up to 200 μl with 10 mm Tris-HCl, PBS, or Elution Buffer provided with this kit. 2. Add 20 µl Proteinase K Solution and 5 µl RNase A (not provided) to the samples. 3. Add 220 µl MSL Buffer and 5 µl Binding Enhancer to the samples and seal the plate with sealing film (not provided). Vortex for 20 seconds. Note: MSL Buffer and Binding Enhancer can be made as master mix. For 96 samples add 22 ml MSL Buffer with 5 ml Binding Enhancer. Add 225 µl to each sample. 9

11 KF with Included Incubator Protocol 4. Press start on KingFisher 96 whole blood protocol and load plates accordingly. 5. During Pause Step: Add 305 μl ethanol and 10 μl Mag-Bind Particles CND to the samples. 6. Remove Plate containing genomic DNA and store at -20 C 10

12 Mag-Bind Protocol for KF ml Mag-Bind Blood DNA Protocol (1-200 μl Blood) for KingFisher ml The procedure below has been optimized for use with FRESH or FROZEN blood samples 1 to 200 μl in volume. Buffy coat can also be used. Materials Supplied by User: KingFisher ml Incubator capable of 70 C Vortexer Multi-channel pipettor Nuclease-free 1.5 ml microcentrifuge tubes KingFisher tube strips KingFisher ml tip comb 100% Ethanol Isopropanol Optional: 10 mm Tris-HCl or PBS Optional: RNase A Before Starting: Set incubator to 70 C Prepare SPM Wash Buffer and QMP Buffer according to the Preparing Reagents section on Page 4 Add buffers to appropriate strips according to the table below Strip Content Reagent Volume A Sample/ Lysate according to protocols below B QMP Buffer 500 µl C SPM Wash Buffer 600 µl D SPM Wash Buffer 600 µl E Elution Buffer 100 µl 1. Add blood sample to a nuclease-free1.5 ml microcentrifuge tube (not provided). Bring the volume up to 200 μl with 10 mm Tris-HCl, PBS, or Elution Buffer provided with this kit. 2. Add 20 µl Proteinase K Solution and 5 µl RNase A (not provided) to the samples. Mix thoroughly by pipetting up and down 10 times. 11

13 Mag-Bind Protocol for KF ml 3. Add 220 µl MSL Buffer and 5 µl Binding Enhancer to the samples and mix by pipetting up and down 10 times. Note: MSL Buffer and Binding Enhancer can be made as master mix. For 15 samples mix 3.63 ml MSL Buffer with 83 µl Binding Enhancer. Add 225 µl to each sample. 4. Incubate at 70 C for 10 minutes. 5. Fill strips according to the table on Page 11 except for Strip A. 6. Add 305 µl ethanol and 10 µl Mag-Bind Particles CND. Vortex to mix. 7. Transfer the sample to Strip A. 8. Press start on KingFisher ml whole blood protocol and load strips according to the table on the previous page. 9. Remove strip containing genomic DNA and store at -20 C. 12

14 Determination of Yield and Quality The total DNA yield can be determined by a spectrophotometer using deionized water, Tris-HCl buffer, or Elution Buffer as blank. DNA concentration is calculated as: [DNA] = (Absorbance260) x (0.05 μg/ μl) x (Dilution factor) The quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm. A ratio of (A 260 /A 280 ) of corresponds to 85-95% purity. 13

15 Troubleshooting Guide Please use this guide to solve any problems that may arise. We hope that it will aid in clearing up any questions for you. If for any reason you need further assistance, please contact our technical support staff at our Toll Free Number at Possible Problems and Suggestions Problem Cause Solution Low DNA yields Incomplete resuspension of magnetic particles Inefficient cell lysis due to inefficient mixing of MSL Buffer and sample SPM Wash Buffer was not prepared correctly Resuspend the magnetic particles by vortexing before use. Make sure the sample is thoroughly mixed with MSL Buffer. Prepare the SPM Wash Buffer by adding ethanol according to instruction. Problem Loss of magnetic beads during operation Inefficient cell lysis due to decrease of activity of Proteinase K Be careful not remove the magnetic beads during the operation. Add more Proteinase K Solution. Solution No DNA eluted SPM Wash Buffer not diluted with ethanol Prepare SPM Wash Buffer as instructed on the label. Problem with downstream applications Cause Insufficient DNA was used Excess DNA was used for downstream application Solution 1. Use more stating material 2. Quantify the purified DNA accurately and use sufficient DNA. Make sure to use correct amount DNA. 14