Saliva DNA Isolation 96-Well Kit - Protocol
Contents
1. 2 minutes Perform a second binding step as outlined in Step 1b Discard the flowthrough Reassemble the 96 Well Plate and the 96 Well Collection Plate 2 DNA Wash a S 2p Apply 500 uL of Wash Solution A to each well of the 96 Well Plate Centrifuge the assembly at maximum speed or 4 000 x RPM 3420 x g for 2 minutes Note Ensure the entire volume of Solution WN has passed through into the 96 Well Collection Plate by inspecting the 96 Well Plate If the entire wash volume has not passed centrifuge for an additional 2 minutes Discard the flowthrough Reassemble the 96 Well Plate and the 96 Well Collection Plate Repeat Step 2a to wash the plate a second time Discard the flowthrough Pat the bottom of the 96 Well Plate dry Reassemble the 96 Well Plate and the 96 Well Collection Plate Centrifuge the assembly at a minimum speed of 4 000 x RPM 3420 x g for 15 minutes in order to completely dry the plate 3 DNA Elution a b c d Stack the 96 Well Plate on top of the 96 Well Elution Plate Add 100 uL of Elution Buffer B to each well of the 96 Well Plate Centrifuge the assembly at a minimum speed of 2 000 x g 800 RPM for 2 minutes followed by 4 000 x RPM 3420 x g for 3 minutes Store purified gDNA at 20 C for a few days or at 70 C for long term storage Troubleshooting Guide Problem Possible Cause Solution and Explanation Ensure that a vacuum pressure of at le2. ast 650 mbar or 25 Insufficient Vacuum in Hg is developed Centrifuge Ensure that the centrifuge remains at room temperature Clogged temperature too throughout the procedure Temperatures below 15 C may Wells in low cause precipitates to form that can cause the wells to clog Plate Ensure that no more than 0 5 mL of lysed saliva is applied The sample is too during each binding step Clogging can be alleviated by large vacuuming or centrifuging for a longer period of time until the lysate passes through the wells of plate Incomplete lysis of Increased Proteinase K incubation time at 55 C may result The yield of cells in increased yields genomic DNA is low cau rah Some saliva samples contain very little DNA This varies from individual to individual based on numerous variables sample is low DNA was not Traces of salt from the binding step may remain in the sample if the column is not washed twice with Wash DNA does washed twice with l Solution A Salt may interfere with downstream not perform Solution A eee well in applications and thus must be washed from the column sahara Ensure that the dry step after DNA wash is performed in Pp Ethanol carryover order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications Related Products Product Saliva DNA Isolation Kit RU45400 Saliva DNA Collection and Preservation Devices 50 RU49000 Saliva DNA C
3. croorganisms are lysed Nonetheless 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 Micropipettors and multichannel pipettes 96 100 ethanol Isopropanol 55 C Incubator Collection Waste Tray for vacuum manifold For Vacuum Format o Vacuum manifold with vacuum pump capable of generating a minimum vacuum of 650 mbar or 25 in Hg o Sealing tape or pads e For Centrifuge Format o Centrifuge with rotor for 96 well plate assembly such as AllSpin Js 5 3 Rotor for Avanti J 26xp centrifuge Beckman Coulter or similar rotor that can hold the stack of the 96 well plate and the 96 Well Collection Plate and that can reach the minimum speed of 4000 rpm 4000xg Flowchart Procedure for Purifying Total DNA using Norgen s Saliva DNA Isolation 96 Well Kit Add Lysis Buffer F to saliva samples Mix Transfer Add Proteinase K Incubate at 55 C for 10 minutes Add Binding Buffer B mix Add isopropanol mix Bind Wash twice with Wash Solution A Dry plate Elute DNA with Elution Buffer B Pure Saliva DNA Procedures For Vacuum Manifold All vacuum steps are performed at room temperature The correct pressure can be calculated using the conversions 1 mbar 100 Pa 0 0394 in Hg 0 75 mm Hg 0 0145 psi For Centrifugation All centrifugation steps are carried out at room temperature in a c
4. g DR Saliva DNA Isolation 96 Well Kit NORGEN BIOTEK wie CORPORATION Product RU35200 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Product Insert Norgen s Saliva DNA Isolation 96 Well Kit provides a rapid method for the high throughput isolation of total genomic DNA from saliva Human genomic DNA extracted from buccal epithelial cells and white blood cells found in saliva is of the highest integrity and can be used in various downstream applications such as real time PCR Southern blotting sequencing macro micro array and SNP assays Purification can be performed on either a vacuum manifold or using centrifugation This product is compatible with Norgen s Saliva DNA Collection and Preservation Kit as well as Norgen s Buccal DNA Collection and Preservation Kit Advantages Sample collection is non invasive and painless Fast and easy high throughput processing using 96 well plates Process using a centrifuge or vacuum manifold DNA can be isolated and detected from as little as 100 uL of saliva Isolate high quality genomic DNA Specifications Kit Specifications Binding Capacity Per Well 50 ug Maximum Saliva Input 0 5 mL of preserved saliva Average Yield from 0 25 mL of Saliva 3 5 ug Average Purity OD 260 280 1 7 Time to Complete 96 Purifications 40 minutes Kit Componen
5. ix by vortexing and incubate at 55 C for 10 minutes Add 200 uL of Binding Buffer B 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 Step 2 Total DNA Isolation 1B Norgen s Saliva DNA Collection and Preservation Device a 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 vortex the saliva sample for 10 seconds Transfer 500 uL of preserved saliva to a clean tube Add 20 uL of Proteinase K Mix by vortexing and incubate at 55 C for 10 minutes Add 200 uL of Binding Buffer B to the saliva sample Mix by vortexing and incubate at 55 C for 5 minutes Add 720 uL of lsopropanol and mix by vortexing for a few seconds Proceed to Step 2 Total DNA Isolation 1C Saliva Sample Collection and Lysis Cotton Swab a b Aliquot 500 uL of Lysis Buffer F into a sterile microcentrifuge tube not provided Gently place one swab in cheek pouch rotate and move the swab head for 30 seconds to collect as much saliva as possible 9 mpe ze Using sterile techniques clip the swab head into the microcentrifuge tube with Lysis Buffer F If possible repeat steps using a second swab Close the lid of the tube Vortex gently and incubate for 5 minutes at room temperature Add 20 uL of Proteina
6. ly up to 750 uL of the lysate into each well of the 96 Well Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough Reassemble the 96 Well Plate and the vacuum manifold Note Ensure that all of the lysate from each well has passed through into the collection waste tray If the entire lysate volume has not passed apply vacuum for an additional 2 minutes c Perform a second binding step as outlined in Step 1a d Turn off vacuum and ventilate the manifold Discard the flowthrough Reassemble the 96 Well Plate and the vacuum manifold 2 DNA Wash a Apply 500 uL of Wash Solution A to each well of the 96 Well Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Ensure the entire volume of Wash Solution A has passed through into the collection waste tray by inspecting the 96 Well Plate If the entire wash volume has not passed apply vacuum for an additional 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough c Reassemble the 96 Well Plate and the vacuum manifold d Repeat Step 2a to wash the plate a second time e Turn off vacuum and ventilate the manifold Discard the flowth
7. ollection Preservation and Isolation Kit RU35700 Buccal DNA Collection and Preservation Kit 33900 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 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 PIRU35200 5 M14
8. onventional laboratory centrifuge using a swing bucket rotor capable of 3000 5000 x g such as Thermo Fisher IEC Centra CL3 series or Beckman GS 15R Notes Prior to Use Ensure that all solutions are at room temperature prior to use Freshly collected preserved or frozen saliva without preservation may be used as a starting material Prepare a working concentration of Wash Solution A by adding 90 mL of 96 100 ethanol to be provided by the user to the supplied bottles containing concentrated Wash Solution A This will give a final volume of 128 mL The labels on the bottles have a box that can be checked to indicate that ethanol has been added The volumes stated in each procedure for lysate preparation are the volumes required to prepare samples for each well of the 96 well plate The maximum recommended input of lysed saliva mixture is 500 uL per well of the 96 Well Plate 1A Saliva Sample Collection and Lysis Spitting a f g 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 wells Ten minutes after rinsing collect saliva by spitting into a sterile collection tube or vial not provided Transfer 250 uL of saliva to a sterile tube and add 250 uL of Lysis Buffer F Vortex the saliva sample for 10 seconds Add 20 uL of Proteinase K M
9. rough Pat the bottom of the 96 Well Plate gently to remove any residual wash buffer Reassemble the 96 Well Plate and the vacuum manifold Apply vacuum for an additional 10 minutes in order to completely dry the plate g Turn off vacuum and ventilate the manifold h Pat the bottom of the 96 Well Plate gently to remove any residual wash buffer 3 DNA Elution a Replace the collection waste tray in the vacuum manifold with the provided 96 Well Elution Plate Complete the vacuum manifold assembly with the 96 Well Plate b Add 100 uL of Elution Buffer B to each well of the plate c Apply vacuum for 5 minutes d Store purified gDNA at 20 C for a few days or at 70 C for long term storage B Total DNA Isolation Using Centrifugation 1 Binding DNA to 96 Well Plate a d e Place the 96 Well Plate on top of a provided 96 Well Collection Plate Note The user should ensure that the assembled 96 Well Plate and the 96 Well Collection Plate stack fits into the rotor without interfering with the centrifugation process Apply up to 750 uL of the mixture into each well of the 96 Well Plate Centrifuge the assembly at maximum speed or 4 000 x g 4 000 RPM for 2 minutes Discard the flowthrough Reassemble the 96 Well Plate and the 96 Well Collection Plate Note Ensure that all of the lysate from each well has passed through into the 96 Well Collection Plate If the entire lysate volume has not passed centrifuge for an additional
10. se K Mix by vortexing and incubate at 55 C for 10 minutes Add 200 uL of Binding Buffer B to the saliva sample Mix by vortexing and incubate at 55 C for 5 minutes Add 720 uL of lsopropanol and mix by vortexing for a few seconds Proceed to Step 2 Total DNA Isolation 1D Buccal Cells Sample Collection and Lysis Cotton Swab a b c d e f g h Aliquot 500 uL of Lysis Buffer F into a sterile microcentrifuge tube not provided Gently brush a sterile single use cotton swab inside the mouth along the cheek Clip the cotton tip where the buccal cells were collected and place into the microcentrifuge tube with Lysis Buffer F If possible repeat steps using a second swab Close the lid of the tube Vortex gently and incubate for 5 minutes at room temperature Add 20 uL of Proteinase K Mix by vortexing and incubate at 55 C for 10 minutes Add 200 uL of Binding Buffer B to the saliva sample Mix by vortexing and incubate at 55 C for 5 minutes Add 720 uL of lsopropanol and mix by vortexing for a few seconds Proceed to Step 2 Total DNA Isolation 2 Total DNA isolation Note The isolation of DNA from saliva can be performed using either a vacuum manifold or centrifugation For purification using vacuum follow the procedure in Section 2A and for purification using centrifugation follow the procedure outlined in Section 2B A Total DNA Isolation Using Vacuum Manifold 1 Binding DNA to 96 Well Plate a App
11. ts Storage Conditions and Product Stability Component Product RU35200 192 preps Lysis Buffer F 100 mL Proteinase K 4AmL Binding Buffer B 40 mL Wash Solution A 2x 38 mL Elution Buffer B 30 mL 96 Well Plate 2 96 Well Collection Plate 2 Adhesive Tape 4 96 Well Elution Plate 2 Product Insert 1 The kit contains ready to use Proteinase K which is dissolved in a specially prepared storage buffer The Proteinase K should be stored at room temperature or 4 C All other solutions should be kept tightly sealed and stored at room temperature 15 25 C These reagents should remain stable for at least 2 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use 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 MSDSs These are available as convenient PDF files online at www norgenbiotek com Binding Buffer B contains 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 Saliva of all human and animal subjects is considered potentially infectious However Norgen s preserved saliva is non infectious for all mi
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