E.Z.N.A.®MicroElute Genomic DNA Kit - Omega Bio-Tek
Contents
1. 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer the sample from Step 11 including any precipitate that may have formed to the MicroElute DNA Mini Column Centrifuge at maximum speed for 1 minute Repeat Steps 13 14 until all the sample has been transferred to the MicroElute DNA Mini Column 16 17 18 19 20 21 22 23 24 25 26 Protocol for Forensic Samples Discard the filtrate and the Collection Tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 21 23 for a second DNA Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the MicroElute DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge2. 15 seconds A precipitate may form at this point it will not interfere with DNA isolation Briefly centrifuge to bring down any liquid from the top of the lid Insert a MicroElute DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube 12 13 14 15 16 17 18 19 20 21 22 23 Protocol for Tissue Samples Transfer the sample from Step 10 including any precipitate that may have formed to the MicroElute DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and the Collection Tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 19 21 for a second DNA Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 minute
3. Semen Spots E Z N A MicroElute Genomic DNA Kit Protocol for Dried Blood Fluids and Semen Spots Dried blood body fluids and semen samples on filter paper can be processed using the following method Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water baths heat blocks or incubators capable of 55 C and 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol Isopropanol Optional sterile deionized water Optional Dithiothreitol DTT Optional 3M NaOH Before Starting 12 Prepare DNA Wash Buffer and HBC Buffer according to the Preparing Reagents section on Page 4 Set water baths heat blocks or incubators to 55 C and 70 C Heat Elution Buffer to 70 C Cut or punch out the blood or other sample spot from the filter paper Tear or cut the filter paper into small pieces and place into a 1 5 mL microcentrifuge tube not provided Note Use 1 3 punched circles 3 mm diameter for each DNA isolation Add 200 uL TL Buffer and 20 uL OB Protease Solution If you are processing semen spots add 20 uL DTT to each sample Incubate at 55 C for 45 60 minutes in a shaking water bath Note If a shaking water bath is not available vortex the sample every 10 20 minutes 10 11 Protocol for Dried Blood Fluids and Semen Spots Briefly centrifuge to bring down any liquid from the top of the
4. a spectrophotometer using deionized water Tris HCl buffer or Elution Buffer as blank DNA concentration is calculated as DNA Absorbance260 x 0 05 ug uL 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 A of 1 7 1 9 corresponds to 85 95 purity Yields vary with both 260 280 amount and type of sample used Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D3096 02 100 mL per bottle 2 Dilute HBC Buffer with isopropanol as follows and store at room temperature Protocol for Tissue Samples E Z N A MicroElute Genomic DNA Kit Protocol for Tissue Samples The following protocol allows for isolation of genomic DNA from up to 10 mg tissue Yields will vary depending on source To purify very small amounts of DNA from a sample such as low volumes of blood lt 10 uL or micro dissected tissues we recommend adding Linear Acrylamide to BL Buffer to enhance the DNA binding ability of the column In most cases adding 5 10 hg 1 2 uL per sample is sufficient Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water baths heat blocks or incubators capable of 55 C and 70 C Shaking water bath capable of 55 C e Vortexer 100 ethanol lsopropanol Optional
5. E Z N A MicroElute Genomic DNA Kit D3096 00 5 preps D3096 01 50 preps D3096 02 200 preps May 2013 E Z N A MicroElute Genomic DNA Kit Table of Contents A A 2 Kit Contents Storage and Stability 3 Determination of DNA Quality and Quantity esssescsesesneeensee 3 Preparing FCO GIVES once 4 Protocol for Tissue Samples ida 5 Protocol for Small Volumes of Blood Serum or Body Fluids 9 Protocol for Dried Blood Body Fluids or Semen Spots 12 Protocol ornato 16 Protocol for Forensic SAMPI S c ccssscsecseesssscsssssessscsessscsnsessssseeseeees 19 Troubleshooting Sulde nennen 23 OO 24 Manual Revision May 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z N A MicroElute Genomic DNA Kit provides a rapid and easy method for the isolation of genomic and mitochondrial DNA from small sample sizes or from a large volume of samples for consistent PCR and other downstream applications This kit can be used for the isolation of genomic DNA from micro dissected tissue cultured cells blood dry blood swabs buffy coat serum urine and plasma The kit allows single or multiple processing of samples There is no need for phenol chloroform extractions and time consuming steps such as precipitation with isopropanol or ethanol are eliminated The E Z N A MicroElute Genomic DNA Kit uses the reversible binding properties of the HiBind matrix a silica based materi
6. RNase A 25 mg mL e Optional sterile deionized water Optional 3M NaOH Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the Preparing Reagents section on Page 4 Set water baths heat blocks or incubators to 55 C and 70 C Heat Elution Buffer to 70 C 1 Mince up to 10 mg tissue and transfer to a 1 5 mL microcentrifuge tube not provided 2 Add 200 uL TL Buffer 3 Add 20 uL OB Protease Solution Vortex to mix thoroughly Protocol for Tissue Samples Incubate at 55 C in a shaking water bath Note If a shaking water bath is not available vortex the sample every 10 20 minutes Lysis time will depend on the amount and type of tissue but is usually less than 3 hours Lysis can proceed overnight Centrifuge at maximum speed 13 000 x g for 2 minutes Transfer the supernatant to a clean 1 5 mL microcentrifuge tube not provided Optional Certain tissues such as liver 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 Add 5 uL RNase A 25 mg mL assuming a sample size of 10 mg and incubate at room temperature for 2 minutes Proceed to Step 7 7 10 11 Add 220 uL BL Buffer Vortex to mix thoroughly Note If Linear Acrylamide is needed add 1 2 uL Linear Acrylamide to 220 uL BL Buffer Incubate at 70 C for 10 minutes Add 220 uL 100 ethanol Vortex at maximum speed for
7. al in combination with the MicroElute spin column technology to allow elution volume as small as 10 uL A specially formulated buffer system allows genomic DNA up to 40 kb to bind to the matrix Samples are lysed under denaturing conditions and then transferred to the MicroElute DNA Mini Columns to bind DNA Cellular debris hemoglobin and other proteins are efficiently eliminated via three quick wash steps High quality DNA is eluted in sterile deionized water or low salt buffer New in this Edition HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user 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 D3096 00 D3096 01 MicroElute DNA Mini Columns Storage and Stability All of the E Z N A MicroElute Genomic DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows OB Protease Solution can be stored at room temperature for up to 12 months For long term storage store OB Protease Solution at 2 8 C During shipment or storage in cool ambient conditions precipitates may form in BL Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Determination of DNA Quality and Quantity The total DNA yield can be determined by
8. and 70 C Heat Elution Buffer to 70 C 1 Cut the sample to small pieces and place into a 2 mL microcentrifuge tube not provided 2 Add 300 uL TL Buffer Vortex to mix thoroughly If you are processing semen stains add 20 uL DTT to each sample 3 Add 20 uL OB Protease Solution Vortex at maximum speed for 30 seconds 4 Incubate mixture at 55 C for 60 minutes in a shaking water bath Note If a shaking water bath is not available vortex the sample every 10 20 minutes Lysis time will depend on the amount and type of sample but is approximately one hour Extend incubation time of hair samples until lysis is complete Lysis can proceed overnight for larger samples of nail clippings 19 10 11 12 Protocol for Forensic Samples Briefly centrifuge to bring down any liquid from the top of the tube Add 320 uL BL Buffer Vortex at maximum speed for 20 seconds Incubate at 70 C for 10 minutes Vortex at maximum speed for 10 seconds several times during incubation Centrifuge at maximum speed for 5 minutes Transfer the cleared supernatant to a new 2 mL microcentrifuge tube not provided Add 320 uL 100 ethanol Vortex at maximum speed for 15 seconds Briefly centrifuge to bring down any liquid from the top of the lid Insert a MicroElute DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 13 14 15 20 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column
9. be Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube Add 500 RL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the Collection Tube Protocol for Small Volumes of Blood Serum or Fluids 16 17 18 19 20 21 22 23 24 25 Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 16 18 for a second DNA Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the MicroElute DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided Add 10 50 uL Elution Buffer or sterile deionized water heated to 70 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit at room temperature for 3 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C 11 Protocol for Dried Blood Fluids and
10. ed for 15 seconds Briefly centrifuge to bring down any liquid from the top of the lid Insert a MicroElute DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 12 13 14 13 16 17 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer the sample from Step 10 including any precipitate that may have formed to the MicroElute DNA Mini Column Centrifuge at maximum speed for 1 minute Repeat Steps 14 16 until all the sample has been transferred to the MicroElute DNA Mini Column Discard the filtrate and the Collection Tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube Add 500 RL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions 17 18 19 20 21 22 23 24 25 26 27 28 29 18 Protocol for Swabs Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 20 22 for a second DNA Wash Buffer wash step Centr
11. emen swabs blood swabs and buccal swabs Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g Nuclease free 2 mL microcentrifuge tubes Water baths heat blocks or incubators capable of 55 C and 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol Isopropanol Optional sterile deionized water Optional Dithiothreitol DTT Optional 3M NaOH Before Starting 16 Prepare DNA Wash Buffer and HBC Buffer according to the Preparing Reagents section on Page 4 Set water baths heat blocks or incubators to 55 C and 70 C Heat Elution Buffer to 70 C Place the swab in a 2 mL microcentrifuge tube Add 600 uL TL Buffer and 20 uL OB Protease Solution Vortex at maximum speed for 30 seconds If you are processing semen swabs add 20 uL DTT to each sample Incubate at 55 C for 60 minutes in a shaking water bath Note If a shaking water bath is not available vortex the sample every 10 20 minutes Briefly centrifuge to bring down any liquid from the top of the tube Add 620 uL BL Buffer Vortex at maximum speed for 20 seconds 10 11 Protocol for Swabs Incubate at 70 C for 10 minutes Vortex at maximum speed for 10 seconds several times during incubation Briefly centrifuge to bring down any liquid from the top of the tube Transfer the cleared lysate to a new 1 5 mL microcentrifuge tube not provided Add 620 uL 100 ethanol Vortex at maximum spe
12. ifuge the empty MicroElute DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the MicroElute DNA Mini Column to a nuclease free 2 mL microcentrifuge tube not provided Add 10 50 uL Elution Buffer or sterile deionized water heated to 70 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit at room temperature for 3 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C Protocol for Forensic Samples E Z N A MicroElute Genomic DNA Kit Protocol for Forensic Samples This protocol is designed for isolation of genomic DNA from forensic samples such as hair cigarette butts nail clippings material stained with blood saliva or semen Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water baths capable of 55 C and 70 C Shaking water bath capable of 55 C Vortexer 100 ethanol lsopropanol Optional Dithiothreitol DTT e Optional sterile deionized water Optional 3M NaOH Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the Preparing Reagents section on Page 4 Set water baths to 55 C
13. must be diluted with isopropanol before use Increase incubation time with TL Buffer An Poor cell Iysis oe overnight incubation may be necessary Incomplete Pulverize starting material as indicated in homogenization liquid nitrogen to obtain a fine powder 100 ethanol was not added before adding Before applying DNA sample to column add TL Buffer and 100 ethanol sample to column Ethanol was not added Dilute DNA Wash Buffer with the indicated to the DNA Wash Buffer volume of 100 ethanol before use 23 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 DNase RNase free microcentrifuge tubes 1 5 mL 500 pk 10 pk cs DNase RNase free microcentrifuge tubes 2 0 mL 500 pk 10 pk cs RNase A 5 mL Proteinase K Solution 10 mL Elution Buffer 100 mL DNA Wash Buffer 100 mL Part Number SSI 1210 00 SSI 1310 00 PDO90 AC116 PDR048 PSO10 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 24
14. ollection Tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 19 21 for a second DNA Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the MicroElute DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided 25 26 27 28 Protocol for Dried Blood Fluids and Semen Spots Add 10 50 uL Elution Buffer or sterile deionized water heated to 70 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit at room temperature for 3 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C 15 Protocol for Swabs E Z N A MicroElute Genomic DNA Kit Protocol for Swabs This protocol is designed for the isolation of genomic DNA from s
15. or capable of 70 C Vortexer lsopropanol 100 ethanol PBS e Optional sterile deionized water e Optional 3M NaOH Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the Preparing Reagents section on Page 4 Set water bath heat block or incubator to 70 C Heat Elution Buffer to 70 C 1 Add 1 100 uL sample the sample must be at room temperature to a 1 5 mL microcentrifuge tube not provided 2 Adjust the sample volume to 100 uL with PBS 3 Add 20 uL OB Protease solution Vortex to mix thoroughly 4 Add 120 uL BL Buffer Vortex to mix thoroughly Protocol for Small Volumes of Blood Serum or Fluids Incubate at 70 C for 10 minutes Add 160 uL isopropanol Vortex at maximum speed for 15 seconds A precipitate may form at this point it will not interfere with DNA isolation Briefly centrifuge to bring down any liquid from the top of the lid Insert a MicroElute DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 11 12 13 14 15 10 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer the sample from Step 7 including any precipitate that may have formed to the MicroElute DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and the Collection Tu
16. s to dry the membrane Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications 24 25 26 27 28 Protocol for Tissue Samples Transfer the MicroElute DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided Add 10 50 uL Elution Buffer or sterile deionized water heated to 70 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit at room temperature for 3 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C Protocol for Small Volumes of Blood Serum or Fluids E Z N A MicroElute Genomic DNA Kit Protocol for Small Volumes of Blood Serum or Fluids This protocol is designed for the rapid isolation of DNA from 1 100 uL blood treated with EDTA citrate or heparin based anticoagulants serum plasma buffy coat saliva and urine To purify very small amounts of DNA from a sample such as low volumes of blood lt 10 uL or micro dissected tissues we recommend adding Linear Acrylamide to BL Buffer to enhance the DNA binding ability of the column In most cases adding 5 10 hg 1 2 uL per sample is sufficient Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water bath heat block or incubat
17. tube Add 220 uL BL Buffer Vortex to mix thoroughly Note If only one punch card is processed add 1 ul Linear Acrylamide to the sample Incubate at 70 C for 10 minutes Vortex at maximum speed for 10 seconds several times during incubation Centrifuge at maximum speed for 5 minutes Transfer the cleared lysate to a new 1 5 mL microcentrifuge tube not provided Note For maximum yield collect any remaining liquid from the paper and transfer the entire sample including paper into an Omega Homogenizer Column not provided Cat HCR003 and centrifuge at maximum speed for 2 minutes to collect all of the lysate Add 220 uL 100 ethanol Vortex at maximum speed for 15 seconds Briefly centrifuge to bring down any liquid from the top of the lid Insert a MicroElute DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 12 13 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer the sample from Step 10 including any precipitate that may have formed to the MicroElute DNA Mini Column Centrifuge at maximum speed for 1 minute 13 14 15 16 17 18 19 20 21 22 23 24 14 Protocol for Dried Blood Fluids and Semen Spots Discard the filtrate and the Collection Tube Transfer the MicroElute DNA Mini Column to a new 2 mL C
18. tube not provided 21 27 28 29 30 22 Protocol for Forensic Samples Add 10 50 uL Elution Buffer or sterile deionized water heated to 70 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit at room temperature for 3 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C Please use this please contact Clogged Column Low DNA yield No DNA eluted Troubleshooting Guide guide to troubleshoot any problems that may arise For further assistance the technical support staff toll free at 1 800 832 8896 Extend incubation time of lysis with TL Buffer and protease Add the correct volume of BL Incomplete lysis Buffer and incubate for specified time at 70 C It may be necessary to extend incubation time by 10 minutes Do not use greater than the recommended Sample too large amount of starting material For larger samples divide into multiple tubes Incomplete Pulverize material as indicated in liquid homogenization nitrogen to obtain a fine powder Clogged column Repeat elution or increase elution volume Poor elution Incubate the column at 70 C for 5 minutes before centrifugation at Follow the protocol closely when adjustin Poor binding to colum eee im y J g the binding conditions DNA Wash Buffer must be diluted with 100 ethanol before use Improper washing z HBC Buffer
Download Pdf Manuals
Related Search