User Manual
Contents
1. Add 200 uL of Binding Solution the saliva sample Mix by vortexing and incubate at 55 C for 5 minutes Add 720 uL of isopropanol and mix by vortexing for a few seconds Proceed to Section 2 Sample Binding to Column Sample Binding to Column Assemble a column with one of the provided collection tubes Apply up to 760 uL of the lysate to the column and centrifuge for 1 minute at 6 000 RPM 3800 x 9 Note Ensure the entire sample has passed through into the collection tube by inspecting the column If the entire sample volume has not passed spin for additional 1 minute c Discard the flowthrough and reassemble the spin column with its collection tube Note Each spin column provided is capable of processing up to 500 uL of lysed saliva If additional DNA isolation is desired use an additional spin column For example if 1 mL of saliva is to be processed use two spin columns and process 500 uL of lysed saliva with each column d Repeat Steps 2b and 2c until all the lysate has passed through the column GO Column Wash Apply 500 uL of Wash Solution to the column and centrifuge for 1 minute at 8 000 RPM 6800 x g 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 D b Apply 500 uL of Wash Solution to the column2. and centrifuge for 1 minute at maximum speed Discard the flowthrough and reassemble the spin column with its collection tube c Spin the column for 2 minutes at maximum speed in order to thoroughly dry the column Discard the collection tube DNA Elution Place the column into a fresh 1 7 mL Elution tube provided with the kit Add 100 uL of Elution Buffer to the column Incubate for 5 minutes at 55 C Centrifuge for 2 minutes at 2 000 RPM 425x g followed by a second spin for 1 minute at Maximum speed gt o aR Note For more concentrated sample 50 uL Elution Buffer can be used 5 Storage of DNA The purified DNA sample may be stored at 4 C for a few days Itis recommended that samples be placed at 20 C for long term storage Related Products Product Saliva DNA Collection and Preservation Devices 50 49000 Saliva DNA Collection Preservation and Isolation Kit 35700 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 Troubleshooting Guide Problem Possible Cause Solution and Explanation Check the centrifuge to ensure that it is capable of generating the required RPMs Sufficient centrifugal force is required to move the liquid phase through the column Also ensure th
3. 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 Saliva DNA Isolation Kit Dx Product Insert Dx45400 CE AR PIDx45400 3 Intended Use Norgen s Saliva DNA Isolation Kit Dx provides a fast and simple procedure for isolating genomic DNA from saliva samples collected and preserved using Norgen s Saliva DNA Collection and Preservation Devices as well as fresh saliva for subsequent in vitro diagnostic use Purification is based on spin column chromatography The genomic DNA is preferentially purified from other cellular components such as proteins and RNA Saliva DNA can either be isolated from saliva samples collected and preserved using Norgen s Saliva Collection and Preservation Devices or fresh saliva samples 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 Saliva DNA Isolation 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 Saliva DNA Isolation Kit Dx is intended for use by professional users such as technicians
4. a Sample Collection and Lysate Preparation Samples collected using Norgen s Saliva DNA Collection and Preservation Devices Collect and preserve saliva samples using Norgen s Saliva DNA Collection and Preservation Devices please see Related Products Table Before using the preserved saliva for DNA isolation mix the preserved saliva by inversion for a few seconds Aliquot 500 uL of preserved saliva to a microcentrifuge tube Add 20 uL of Proteinase K vortex before use Mix by vortexing and incubate at 55 C for 10 minutes Add 200 uL of Binding Solution to the saliva sample Mix by vortexing and incubate at 55 C for 5 minutes Add 720 uL of Ilsopropanol and mix by vortexing for a few seconds Proceed to Section 2 Sample Binding to Column B Fresh Saliva Samples a a0 NO TD Prior to collection of saliva samples the donor should rinse their mouth with a few millilitres of water for 10 seconds in order to remove any food particles that may be present If food particles are present they may cause clogging of the column Ten minutes after rinsing collect saliva by spitting into a sterile collection tube or vial not provided The amount of saliva collected should be at least 100 uL but not more than 2 mL Transfer 250 uL of liquid saliva to a sterile microcentrifuge tube Add 250 uL Lysis Solution and mix by vortexing Add 20 uL of Proteinase K vortex before use Mix by vortexing and incubate at 55 C for 10 minutes
5. an be used until the expiration date specified on their labels Precautions Ensure that 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 MSDS Ss These are available as convenient PDF files online at www norgenbiotek com The Binding Solution contains guanidinium salts and should be handled with care Guanidinium salts forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Saliva 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 saliva Customer Supplied Reagents and Equipment e Benchtop microcentrifuge Micropipettors Water for rinsing mouth Norgen s Saliva DNA Collection and Preservation Devices optional 96 100 ethanol lsopropanol 55 C Incubator Procedure All centrifugation steps are carried out at room temperature in a benchtop microcentrifuge that has a maximum speed 20 000 x g approximately 14 000 rom Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds Flow Chart Procedure for Purifying Saliva DNA using Norgen s Saliva DNA Isolation Kit Dx Preserved Saliva Samples col
6. at the correct spin times are followed Spinning for a few additional minutes will help Centrifugation speed was too low or spin time was inadequate The micro spin Too many cells were applied to the column column is Ensure that no more than 0 5 mL of preserved clogged The sample is too large saliva is applied to the column Clogging can be alleviated by centrifuging for a longer period of time until the lysate passes through the column The lysate binding To ensure a homogeneous solution vortex for 10 solution mixture Is not 15 seconds before applying the lysate to the spin homogeneous column incomblete Wee orcalle Increased Proteinase K incubation time at 55 C p y may result in increased yields The DNA elution is Perform an additional centrifugation of 2 minutes at The yield of incomplete 14 000 x g to ensure that all the DNA is eluted genomic DNA i Some saliva samples contain very little DNA This DNA concentration in varies from individual to individual based on the saliva sample being numerous variables Increased proteinase K used is low incubation time at 55 C may result in increased yields Traces of salt from the binding step may remain in DNA was not washed the sample if the column is not washed with the with the provided Wash Wash Solutions Salt may interfere with DNA does not Solutions downstream applications and thus must be perform well in washed from the column downstream applications E
7. enced and trained in molecular biological techniques including DNA isolation Norgen s Saliva DNA Isolation 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 Saliva DNA Isolation 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 Corp 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 PIDx45400 3
8. lected using Norgen s Saliva DNA Collection and Preservation Devices or a mixture of fresh saliva and Lysis Solution Mix Transfer Add Proteinase K incubate at 55 C for 10 minutes Add Binding Solution incubate at 55 C for 5 minutes Add isopropanol mix f Bind SPIN DAL Wash twice with Wash Solution Dry Spin SPIN DAL Elute DNA with Elution Buffer SPIN 3 Pure Saliva DNA Notes prior to use 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 Saliva samples should be collected and preserved using Norgen s Saliva DNA Collection and Preservation Devices please see Related Products Table e Prepare a working concentration of Wash Solution by adding 42 mL of 96 100 ethanol to be provided by the user to the supplied bottle containing concentrated Wash Solution This will give a final volume of 60 mL The label on the bottle has a box that can be checked to indicate that ethanol has been added e The protocol provided is for isolation of DNA from 500 uL of preserved saliva If a starting volume of less than 250 uL is preferred scale down the amount of Proteinase K Binding Buffer B and Ilsopropanol used in Section 1 proportionally e All centrifugation steps are performed at room temperature e Always vortex the Proteinase K before use Saliv
9. nsure 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 Carry out a digestion with RNase A on the elution if the RNase present will interfere with downstream applications Refer to manufacturer s instructions regarding amount of enzyme to use optimal incubation time and temperature RNA is present RNA is coeluted with the in eluted DNA DNA Product Use Restriction Norgen s Saliva DNA Isolation Kit Dx provides a fast and simple procedure for isolating genomic DNA from saliva samples collected and preserved using Norgen s Saliva DNA Collection and Preservation Devices as well as fresh saliva for subsequent in vitro diagnostic use 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 Saliva DNA Isolation 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 Saliva DNA Isolation Kit Dx is intended for use by professional users such as technicians physicians and biologists experi
10. physicians and biologists experienced and trained in molecular biological techniques including DNA isolation Norgen s Saliva DNA Isolation 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 45400 50 samples o R Ny a fo Cl oy not Use ES 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 Sample collection is non invasive and painless Fast and easy processing using a rapid spin column format Isolate high quality genomic DNA Compatible with preserved saliva samples collected using Norgen s Saliva DNA Collection and Preservation Devices please see Related Products Table Specifications Kit Specifications Maximum Saliva Inout 0 5 mL preserved saliva j 0 25 mL fresh saliva Average Yield from 0 25 mL of Saliva Average purity OD260 280 1 7 2 1 Time to Complete 10 Purifications Average DNA yield will vary depending on the donor Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature All solutions and plastics c
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