E.Z.N.A.®HP Tissue DNA Maxi Kit - Omega Bio-Tek
Contents
1. F OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A HP Tissue DNA Maxi Kit D5196 00 2 preps D5196 01 10 preps April 2013 For research use only Not intended for diagnostic testing E Z N A HP Tissue DNA Maxi Kit Table of Contents Introduction and OVEFVIEW csccssscsscsecssscssecsecnseeseecneceneersees Kit Contents Storage and Stability secsecsssecsecseeeneers Preparing REAGCINS visissisissnanieansiaenaivanuninanmnen E Z N A HP Tissue Maxi Kit Protocol ccssssessssseessseseeeees Troubleshooting Guide sessssssssssessesssssssssessceessesssssssseeeeeee Manual Revision April 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A HP Tissue DNA Maxi Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from tissue samples up to 2 grams in size The specially formulated buffer system ensures the optimal lysis of tissues rich in fat polysaccharides and fibers such as brain adipose and muscle This kit can also isolate DNA from molluscs insects arthropods roundworms flatworms and other invertebrate tissue samples rich in mucopolysaccharides The procedure relies on the well established properties of the cationic detergent cetyltrimethyl ammonium bromide CTAB in conjunction with the selective DNA binding of Omega Bio tek s HiBind matrix Samples are homogenized and lysed in a high2. 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 Increase incubation time with MTL1 Buffer and Proteinase K Solution An overnight incubation may be necessary Centrifuge to remove any insoluble particles Incomplete lysis Clogged Column Do not use greater than the recommended Sample too large amount of starting material For larger samples divide into multiple tubes Incomplete Pulverize starting material as indicated in homogenization liquid nitrogen to obtain a fine powder Ceegedcotume_ see2b elution or increase elution volume Incubation of column at 70 C for 5 minutes with dH O or Tris buffer prior to centrifugation may increase yields Poor elution No DNA eluted Follow the protocol closely when adjusting the binding conditions Adjust volumes of BL Buffer and ethanol in proportion norovei washin DNA Wash Buffer must be diluted with 100 prop g ethanol as specified on Page 4 before use Resin from the column may be present in eluate Avoid centrifugation at speeds higher than specified The material can be removed from the eluate by centrifugation It will not interfere with PCR or restriction digests Poor binding to column Extended centrifugation during elution step LOW A eof Aaso Increase incubation time with MTL1 Buffer ratio R Poor cell lysis and Protei
3. les should be used for Southern analyses 1 Pulverize 500 mg tissue in liquid nitrogen with mortar and pestle and transfer the powder in a clean 50 mL centrifuge tube Sample can also be ground and homogenized by beads mill Note The amount of starting material depends on the sample and can be increased if acceptable results are obtained with the suggested 500 mg tissue In any event use no more than 2 g tissue per HiBind DNA Maxi Column as DNA binding capacity 2 5 mg may be exceeded Difficult tissues may require starting with less than 500 mg tissue and doubling all buffer volumes to ensure adequate lysis 2 Add9mLMTL1 Buffer and 300 uL Proteinase K Solution Vortex to mix thoroughly E Z N A HP Tissue DNA Maxi Kit Protocol 3 Incubate at 60 C for a minimum of 2 hours or until the entire sample is solubilized Note Actual incubation time varies and depends tissue type Most samples require no more than 4 hours Alternatively an overnight incubation at 55 C will produce adequate results 4 Add 9 mL chloroform isoamyl alcohol 24 1 Vortex at maximum speed for 15 seconds 5 Centrifuge at 4 000 x g for 5 minutes at room temperature 6 Carefully transfer the upper aqueous phase to a clean 50 mL centrifuge tube Avoid the milky interface containing contaminants and inhibitors In most cases 6 mL upper phase can be transferred Note This step will remove much of the polysaccharides and proteins from solution and imp
4. mponents are guaranteed for at least 12 months from the date of purchase when stored as follows RNase A must be stored at 2 8 C Proteinase K Solution can be stored at room temperature for 12 months For long term storage gt 12 months store at 2 8 C Store all other components at room temperature 22 25 C Check buffers for precipitates before use Redissolve any precipitates by warming to 37 C Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D5196 01 120 mL E Z N A HP Tissue DNA Maxi Kit Protocol E Z N A HP Tissue DNA Maxi Kit Protocol Materials and Equipment to be Supplied by User e Centrifuge equipped with swing bucket rotor capable of 5 000 x g Sterile 50 mL centrifuge tubes Water bath capable of 60 C Mortar and pestle Liquid nitrogen 100 ethanol Sterile deionized water Chloroform e lsoamyl alcohol Optional RNase A 25 mg mL e Optional 3M NaOH Before Starting e Prepare DNA Wash Buffer according to the directions in the Preparing Reagents section on Page 4 Prepare chloroform isoamyl alcohol 24 1 Heat water bath to 60 C Samples preserved in formalin should be rinsed in xylene and then ethanol before processing Note that results obtained with formalin fixed tissues generally depend on the age and size of the specimen Purified material is usually adequate for PCR amplification but fresh or frozen samp
5. nase K Solution An overnight incubation may be necessary Following Step 13 wash the column with a mixture of 300 uL BL Buffer and 300 uL 100 ethanol before proceeding to Step 14 Trace protein contaminants remain Troubleshooting Guide Increase incubation time with MTL1 Buffer Poor cell lysis and Proteinase K Solution An overnight incubation may be necessary Incomplete Pulverize starting material as indicated in homogenization liquid nitrogen to obtain a fine powder 100 ethanol No DNA eluted not added before transferring sample to column Before transferring the sample to the column add BL Buffer and 100 ethanol as indicated in Steps 7 9 100 ethanol was not added to DNA Wash Buffer Dilute DNA Wash Buffer with the indicated volume of 100 ethanol before use 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 10
6. on tube Repeat Steps 10 12 until all the remaining sample has been transferred to the HiBind DNA Maxi Column Add 10 mL HB Buffer Centrifuge at 4 000 x g for 5 minutes at room temperature Discard the filtrate and reuse the collection tube Add 15 mL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Centrifuge at 4 000 x g for 5 minutes at room temperature Discard the filtrate and reuse the collection tube Repeat Steps 17 19 for a second DNA Wash Buffer wash step 21 22 23 24 25 26 27 E Z N A HP Tissue DNA Maxi Kit Protocol Centrifuge the empty HiBind DNA Maxi Column at 4 000 x g for 10 minutes at room temperature to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the HiBind DNA Maxi Column to a clean 50 mL centrifuge tube Add 2 4 mL Elution Buffer 10 mM Tris buffer pH 8 5 or water heated to 60 C directly onto the center of HiBind DNA Maxi Column matrix Let sit for 2 5 minutes at room temperature Centrifuge at 4 000 x g for 5 minutes at room temperature Repeat Steps 23 25 for a second elution step Store DNA at 20 C Note Typically a total of 400 ug DNA with absorbance ratio A A Of 1 7 1 9 can be obtained from 500 mg animal tissue Yields vary depending on source and quantity of starting material used
7. rove spin column performance downstream If little of the upper aqueous phase separates add 2 mL MTL1 Buffer and vortex to mix Centrifuge as above and transfer the upper aqueous solution into a new tube Optional Certain tissues such as liver tissue have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 1 Add 30 uL RNase A 25 mg mL per 500 mg tissue 2 Let sit at room temperature for 5 minutes 3 Proceed to Step 7 below 7 Add equal volume BL Buffer Vortex to mix thoroughly Note A wispy precipitate may form upon the addition of BL Buffer This is does not interfere with DNA recovery 8 Incubate at 70 C for 10 minutes 9 Add equal volume 100 ethanol Vortex at maximum speed for 30 seconds Note If precipitation can be seen at this point break the precipitation by passing through a needle and syringe E Z N A HP Tissue DNA Maxi Kit Protocol Optional Protocol for Column Equilibration 10 11 12 13 14 15 16 17 18 19 20 1 Add 3 mL 3M NaOH to the HiBind DNA Maxi Column 2 Centrifuge at 4 000 x g for 3 minutes 3 Discard the filtrate and reuse the collection tube Transfer the sample from Step 9 including any precipitation that may have formed to a HiBind DNA Maxi Column Centrifuge at 4 000 x g for 5 minutes at room temperature Discard the filtrate and reuse the collecti
8. salt buffer containing CTAB and digested with proteinase After addition of chloroform the homogenate is separate into aqueous and organic phases by centrifugation The upper aqueous phase is extracted and BL Buffer is added to provide appropriate binding conditions The sample is then transferred to the HiBind DNA Maxi Column where the genomic DNA binds to the membrane and salt and other contaminants are efficiently washed way High quality genomic DNA is eluted with Elution Buffer or water Purified DNA is suitable for most downstream applications such as endonuclease digestion thermal cycle amplification and hybridization techniques Each HiBind DNA Maxi column can bind approximately 2 5 mg genomic DNA Using greater than 2 g tissue or 5 x 108 cells is not recommended 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 resuspension step to 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 e Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents aa oeo oen Storage and Stability All E Z N A HP Tissue DNA Maxi Kit co
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