Mag-Bind® Tissue DNA KF 96 Kit - Omega Bio-Tek
Contents
1. well magnet KingFisher 96 deep well plates KingFisher 96 plates Tip sleeve for KingFisher 96 deep well magnet Sealing film Optional E Z 96 Lysate Clearance Plate Cat FL9601 Before Starting Set Incubator to 56 C Prepare Reagents according to Page 4 1 Mince up to 10 mg of tissue or two pieces of mouse tail 0 2 0 5 cm in length into a 96 well deep well plate 2 Add 250 LTL Buffer Note Cut the tissue into small pieces to speed up lysis 3 Add 20 uL Proteinase K Solution Seal the plate with sealing film Vortex to mix thoroughly 4 Incubate at 56 C for 1 3 hours in a shaking water bath Note If a shaking water bath is not available incubate the plate in an incubator and vortex the plate every 20 30 minutes Lysis time depends on the amount and type of tissue but is usually less than 3 hours Lysis can proceed overnight Mag Bind Tissue DNA KF 96 Kit Protocols Important Some samples may contain tissues types such as hair or cartilage that can not be completely digested with Proteinase K Those tissues will interfere with the DNA binding and bead collection causing lower DNA yield and quality To ensure that the tissue lysate is completely free of those tissues it is strongly recommended to use Omega Bio tek s E Z 96 Lysate Clearance Plate Cat FL9601 as follows below Place a Lysate Clearance Plate on a new KingFisher 96 deep well plate Transfer the tissue lysate from Step 4 to the Kin2. Ca OMEGA Innovations in nucleic acid isolation Product Manual Mag Bind Tissue DNA KF 96 Kit M6329 00 1 x 96 preps M6329 01 4 x 96 preps M6329 02 20 x 96 preps July 2012 For research use only Not intended for diagnostic testing Mag Bind Tissue DNA KF 96 Kit Table of Contents Introduction and OVEFVIEW cscssccsscsscsseccecssecnseessecneceneerses 2 Kit Contents Storage and Stability secsessseeceecseeeners 3 Preparing Reagents essseesssseessseerssseresseeessscesssseosseeossesosseseossss 4 KingFisher Pipetting INStructions ssessssccecseesseessecneeseers 5 Recommended ProgramM ssesssssssseseesesssssseeeseesssssssssesseesressssssesse 6 Whole Tissue ProtoCol sssssssssseseseesssssssseesseesssssssssesseessessnsssesse 7 Cultured Cell Protocol sssssssssseesereereerserseeeersseseeseeeeeeresesresses 9 Troubleshooting Guide ssssssssseeesssessssssteesersssssssseeeserssesssss 11 POSTING eenia rE E AANE 12 Manual Revision July 2012 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The Mag Bind Tissue DNA KF 96 kit is designed for rapid and reliable isolation of high quality genomic DNA from a wide variety of tissues and cultured cells Up to 10 mg animal tissue or 5 x 10 cells can be processed in less than 1 hour The Mag Bind magnetic beads technology provides high quality DNA which is suitable for direct use in most do
3. arting material Make sure the sample is thoroughly mixed with MSL Buffer Problem with Insufficient DNA was used Quantify the purified DNA accurately and use sufficient DNA applications e DNA a Xcess was used for ny Make sure to use correct amount DNA downstream application downstream 11 12 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license
4. gFisher 96 deep well plate Centrifuge at 2 500 x g at room temperature for 2 minutes Proceed to Step 5 WN 5 Centrifuge at 3 000 x g for 5 minutes at room temperature 6 Transfer 200 uL cleared lysate into a new KingFisher 96 deep well plate Continue to step 4 Note Due to the variation of the water contents of sample the volume of tissue lysate will vary Always check the volume of the cleared lysate from KingFisher 96 deep well plate to ensure the lysate volume is around 200 uL If the volume of cleared lysate is significantly greater or less than 200 uL adjust the volume of MSL Buffer and ethanol proportionally in the downstream steps For example for 220 uL tissue lysate use 220 uL MSL Buffer and 300 uL ethanol Optional Add 5 uL RNase A 25 mg mL assuming a sample size of 20 mg and incubate at room temperature for 2 minutes Proceed to Step 7 7 Add 200 uL MSL Buffer Vortex to mix thoroughly Note If the expected DNA yield is lt 1 ug add 10 uL Binding Enhancer 8 Add 275 uL 100 ethanol and 10 uL Mag Bind Particles CND Note Vortex Mag Bind Particles CND for 3 minutes before adding to the sample or mastermix below 9 Press start on KingFisher 96 Tissue Protocol and load plates accordingly See Pipetting Instructions on Page 5 Mag Bind Tissue DNA KF 96 Kit Protocols Mag Bind Tissue DNA KF 96 Kit Cultured Cell Protocol This protocol is designed for isolation of up to 25 ug genomic DNA fro
5. l the plate with sealing film Vortex to mix thoroughly Add 200 uL MSL Buffer Note If the expected DNA yield is lt 1 ug add 10 uL Binding Enhancer Optional Add 5 uL RNase A 25 mg mL and incubate at room temperature for 2 minutes Proceed to Step 8 5 10 Press start on KingFisher 96 Tissue Protocol and load plates accordingly See Pipetting Instructions Page 6 During the Pause Step add 275 uL 100 ethanol and 10 uL Mag Bind Particles CND Note Vortex Mag Bind Particles CND for 3 minutes before adding to the sample A mastermix of ethanol and Mag Bind Particles CND can be prepared prior to this step Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Incomplete re suspension Resuspend the magnetic particles by of magnetic particle vortexing before use Inefficient cell lysis due to inefficient mix of MSL Buffer and sample Low DNA SPM Wash Buffer was not Prepare the SPM Wash Buffer by adding yields prepared correctly ethanol according to instruction Loss of magnetic beads Be careful not to remove the magnetic during operation beads during the operation Inefficient cell lysis due to decreased activity of Add more Proteinase K Proteinase K No DNA SPM Wash Buffer was not Prepare SPM Wash Buffer as instructed eluted diluted with ethanol on Page 4 Use more st
6. long term storage gt 12 months store Proteinase K Solution at 2 8 C Binding Enhancer and Mag Bind Particles CND must be stored at 2 8 C Under these conditions performance of all components of the kit are guaranteed at least 12 months Under cool ambient conditions a precipitate may form in the MSL and TL Buffers In case of such an event heat the bottle at 37 C to dissolve the precipitate Preparing Reagents 1 Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature M6329 01 336 mL M6329 02 700 mL per bottle 2 Dilute QMP Buffer with isopropanol as follows and store at room temperature M6329 01 120 mL M6329 02 300 mL per bottle KingFisher Pipetting Instructions Pipetting Instructions for KingFisher 96 and Mag Bind Tissue DNA KF 96 protocols O rme Plate content Reagent votum a o owe A KingFisher 96 well deep well plate B KingFisher 96 KF plate When using samples of Spleen Kidney Liver or Lung tissues Elution Buffer should be adjusted to 200 400 uL for optimal results Prepare sample lysate by following the instructions based on sample type Add 500 uL QMP Buffer to Plate 2 Add 600 uL SPM Wash Buffer to Plate 3 Add 600 uL SPM Wash Buffer to Plate 4 Add 100 ul Elution Buffer to Plate 5 Recommended Program The Following recommendation are based upon Thermo Fisher s KingFisher Software Version 2 6 2 Contact the instrument s manufacturer for
7. m 5 x 10 cultured cells To increase throughput the incubation step at 55 C for 10 minutes performed on the KingFisher can be performed externally Contact Omega Bio tek for a modified protocol for the KingFisher instrument Materials and Reagents to be Supplied by User 100 Ethanol PBS 4 C e 8 or 12 channel pipette Reagent reservoirs e KingFisher 96 or KingFisher Flex 96 with deep well magnet Deep well KingFisher 96 plates KingFisher 96 plates Tip sleeve for KingFisher 96 deep well magnet Sealing film Before Starting Prepare Reagents according to Page 4 1 Prepare the cell suspension using one of the following depending on the starting material Frozen cell samples Thaw cells Pellet the cells Wash the cells with cold 4 C PBS Resuspend cells in 180 uL cold PBS Proceed to Step 2 VU kwWn gt For cells grown in suspension Harvest 5 x 108 cells Centrifuge at 1 200 x g Discard the supernatant Wash the cells with cold 4 C PBS Resuspend cells in 180 uL cold PBS Proceed to Step 2 Au PWN gt Mag Bind Tissue DNA KF 96 Kit Protocols For cells grown in a monolayer 1 Harvest the cells by either using a trypsin treatment or scraping with rubber policemen 2 Wash the cells with cold 4 C PBS Resuspend cells in 180 uL cold PBS 4 Proceed to Step 2 Transfer the sample to a KingFisher 96 deep well plate Add 20 uL Proteinase K Solution Sea
8. 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 Plate Ethanol Wash 1 Mag Bind Tissue DNA KF 96 Kit Protocols Mag Bind Tissue DNA KF 96 Kit Whole Tissue Protocol Materials and Reagents to be Supplied by User Centrifuge capable of at least 3 000 x g Rotor adapter for 96 well microplates Shaking water bath or incubator capable of 56 C 100 Ethanol e 8 or 12 channel pipette Reagent reservoirs e KingFisher 96 or KingFisher Flex 96 with deep
9. wnstream applications such as amplifications and enzymatic reactions This kit can be easily adapted with automated system and the procedure can be scaled up or down allowing purification from various amounts of starting materials Overview If using the Mag Bind Tissue DNA 96 KF Kit for the first time please read this booklet to become familiar with the procedure and its various modification Animal tissue or culture cells are pre treated with proteinase K and then lysed in a specially formulated buffer containing detergent DNA was bound to the surface of Mag Bind magnetic particles Proteins polysaccharides and cellular debris are efficiently washed a 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 This manual has been edited for content and redesigned to enhance user readability Proteinase K is now supplied in a liquid form eliminating the step to resuspend prior to use Proteinase K Solution can be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Kit Contents Mag Bind Particles CND SPM Buffer Proteinase K Solution Storage and Stability Most components of the Mag Bind Tissue DNA KF 96 Kit can be stored at room temperature Proteinase K Solution can be stored at room temperature for 12 months For
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