User Manual
Contents
1. 4c Collect second elution into a new microcentrifuge tube The yield can be improved by an additional 20 30 when this second elution is performed 2079 5 Note A smaller elution volume down to 50 uL can be used to obtain a more concentrated sample For maximum yield 200 uL elutions should be used Relative Recovery from 2 Elutions using Different Elution Volumes Relative Concentration of the First Elution using Different Elution Volumes Relative concentration 100 0 5 Storage of DNA The purified DNA sample may be stored at 4 C for a few days It is recommended that samples be placed at 20 C for long term storage Troubleshooting Guide Problem Possible Cause Solution and Explanation Check Protease K activity Also ensure that correct Inefficient cell lysis volume of Lysis Solution was added to the blood sample When a high cell number is expected in the blood sample ensure that the optional spin for 2 minutes at 14 000 rom after the Proteinase K incubation is performed Take the clean supernatant only for the next binding step Cell debris may be The spin clogging the column column Is clogged Too many cells were applied to the column Ensure that Proteinase K and Lysis Solution are proportionally added as the blood volume is increased Clogging can be alleviated by centrifuging for a longer period of time until the lysate passes through the column The sample is too large Ensure that correct v2. Genomic DNA Isolation Mini Kit Dx in conjunction with an in vitro diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications should be used Norgen s Blood Genomic DNA Isolation Mini Kit Dx is intended for use by professional users such as technicians physicians and biologists experienced and trained in molecular biological techniques including experience with whole blood samples and DNA isolation Norgen s Blood Genomic DNA Isolation Mini Kit Dx does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay The respective user is liable for any and all damages resulting from application of Norgen s Blood Genomic DNA Isolation Mini Kit Dx for use deviating from the intended use as specified in the user manual All products sold by Norgen Biotek are subjected to extensive quality control procedures and are warranted to perform as described when used correctly Any problems should be reported immediately The kit contents are for laboratory use only and they must be stored in the laboratory and must not be used for purposes other than intended The kit contents are unfit for consumption Authorized Representative EMERGO EUROPE Molenstraat 15 2513 BH The Hague The Netherlands em Norgen Biotek Cor
3. a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com The Lysis Solution and Wash Solution I contain guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with blood Customer Supplied Reagents and Equipment e Benchtop microcentrifuge Micropipettors 2 mL microcentrifuge tubes 96 100 ethanol 55 C waterbath or incubator Procedure All centrifugation steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM RCF 1 118 x 105 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary
4. of 42 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Prepare a working concentration of the Wash Solution II by adding 42 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution Il This will give a final volume of 60 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Always vortex the Proteinase K before use Sample Preparation Add 20 uL of Proteinase K to a microcentrifuge tube Transfer 20 200 uL of blood sample to the tube containing Proteinase K Add 300 uL of Lysis Solution to the blood and mix well by vortexing for 10 seconds Briefly spin the tube to collect any drops of liquid from the inside of the lid Incubate at 55 C for 10 minutes Optional If any debris is present in the sample centrifuge for 2 minutes at 14 000 x g 14 000 RPM to precipitate Transfer the clean supernatant to a microcentrifuge tube prior to Step g Briefly spin the tube to collect any drops of liquid from the inside of the lid Add 250 uL of 96 100 Ethanol to the sample and mix well by vortexing for 10 seconds Briefly spin the tube to collect any drops of liquid from the inside of the lid Sample Binding to Column Assemble a column with one of the provided collection tubes Apply the lysate to the column and centrifuge for 1 minute at 6 000 x g 8 000 RPM Discard the flowthro
5. R 3430 Schmon Parkway N O E N Thorold ON Canada L2V 4Y6 Phone 905 227 8848 BIOTEK a CORPORATION Fax 905 227 1061 Email techsupport norgenbiotek com Blood Genomic DNA Isolation Mini Kit Dx Product Insert Dx46300 CE IVD AR PIDx46300 2 Intended Use Norgen s Blood Genomic DNA Isolation Mini Kit Dx is designed for the rapid preparation of genomic DNA from up to 200 uL of whole blood for subsequent in vitro diagnostic use Both fresh and frozen anticoagulated blood may be used with this procedure Purification is based on spin column chromatography as the separation matrix Norgen s column binds DNA under optimized salt concentrations and releases the bound DNA under low salt and slightly alkali conditions This kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated using the DNA isolated with Norgen s Blood Genomic DNA Isolation Mini Kit Dx in conjunction with an in vitro diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications should be used Norgen s Blood Genomic DNA Isolation Mini Kit Dx is intended for use by professional users such as technicians physicians and biologists experienced and trained in molecular biological techniques i
6. g force Flow Chart Procedure for Purifying Blood DNA using Norgen s Blood Genomic DNA Isolation Mini Kit Dx Obtain anticoagulated blood sample and transfer into a tube containing Proteinase K 7 Add Lysis Solution Vortex Spin briefly Incubate N 3 optional r SPI Add Ethanol A Bind to column SPIN Ae Wash once with Wash Solution l Wash twice with Wash Solution II Dry spin SPIN et Elute DNA with Elution Buffer SPIN ay Pure Genomic DNA Notes prior to use 1 a b C d e f ze OFN 2 e Ensure that all solutions are at room temperature prior to use and that no precipitates have formed If necessary warm the solutions and mix well until the solutions become clear again e A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed e For best results the use of whole blood collected into tubes containing an anticoagulant is highly recommended e Both fresh and frozen anticoagulated blood may be used with this procedure Ensure that frozen blood is thawed at room temperature prior to starting the protocol e Prepare a working concentration of the Wash Solution I by adding 24 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution I This will give a final volume
7. ncluding experience with whole blood samples and DNA isolation Norgen s Blood Genomic DNA Isolation Mini Kit Dx does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay Kit Components Component Product Dx46300 50 samples 12 mL Spin Columns a Collection Tubes Elution Tubes Product Insert Label Legend Q a boR at m A Do not Use by Batch Catalogue Contains Manu In Vitro Consult Temper reuse Code Number sufficient facturer Diagnostic instructions ature for lt n gt Medical for use limitation tests Device Made in Canada Norgen Biotek Advantages e CE IVD marked in accordance with EU Directive 98 79 EC Fits into in vitro diagnostic workflows Fast and easy processing using a rapid spin column format Isolate high quality genomic DNA free from RNA contamination Recovered genomic DNA is compatible with various downstream applications Specifications Kit Specifications Maximum Blood Input 200 uL Average Yield 200 uL of blood 4 12 ug Time to Complete 10 Purifications Yield will vary depending on the type of blood processed Column Binding Capacity Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature All solutions and plastics can be used until the expiration date specified on their labels Precautions Ensure that
8. olume of Lysis Solution was The yield of Inefficient cell lysis added to blood sample Also increase incubation genomic DNA time up to 15 minutes at 55 C is low Low DNA binding Ensure Ethanol is added to the sample DNA was not washed with the provided Wash DNA does not Solution perform well in downstream applications Ensure the column was washed once with Wash Solution I and twice Wash Solution Il Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications Ethanol carryover Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com Product Use Restriction Norgen s Blood Genomic DNA Isolation Mini Kit Dx is designed for the rapid preparation of genomic DNA from up to 200 uL of whole blood for subsequent in vitro diagnostic use Both fresh and frozen anticoagulated blood may be used with this procedure This kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated using the DNA isolated with Norgen s Blood
9. p 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2014 Norgen Biotek Corp PIDx46300 2
10. ugh Reassemble the column and the collection tube Note Ensure that all of the lysate has passed through into the collection tube If the entire lysate volume has not passed centrifuge for an additional 2 minutes Column Wash Apply 500 uL of Wash Solution I ensure ethanol was added to the column and centrifuge for 1 minute at 6 000 x g 8 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Apply 500 uL of Wash Solution Il ensure ethanol was added to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube c Wash column another time by adding 500 uL of Wash Solution Il and centrifuging for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube d Spin the column for 2 minutes in order to thoroughly dry the column at 14 000 x g 14 000 RPM Discard the collection tube DNA Elution Place the column into a provided 1 7 mL elution tube Add 200 uL of Elution Buffer to the column Incubate at room temperature for 1 minute Centrifuge for 1 minute at 6 000 x g 8 000 RPM Optional An additional elution may be performed if desired by repeating steps 4a
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