Protocol (96-well) - Norgen Biotek Corp.
Contents
1. Component Contents Slurry B1 2x22 mL Lysis Buffer A 2x30 mL Wash Solution A 2 x 38 mL Elution Buffer B 30 mL 96 Well Filter Plate 1 Adhesive Tape 1 96 Well Collection Plate 1 96 Well Elution Plate 2 Product Insert 1 Customer Supplied Reagents and Equipment e For Vacuum Format o Vacuum manifold with vacuum pump capable of generating a minimum pressure of 650 mbar or 25 in Hg Such as Whatman UniVac 3 Vacuum to Collect Manifold o Sealing tape or pads e For Centrifuge Format o Centrifuge with rotor for 96 well plate assembly such as Thermo Fisher IEC Centra CL3 series or Beckman GS 15R Centrifuge with a swinging bucket rotor capable of 2000 RPM Micropipettors 96 100 ethanol 60 C incubator 15 mL tubes 50 mL tubes Lysozyme if bacterial gDNA isolation is needed Proteinase K 20 mg mL Optional Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25 C for up to 1 year without showing any reduction in performance It is recommended to warm up Slurry B1 and Lysis Buffer A for 20 minutes at 60 C if any salt precipitation is observed Slurry B1 contains a grey resin that will not dissolve when warmed Quality Control In accordance with Norgen s Quality Management System each lot of Norgen s Urine DNA Isolation 96 Well Kit Slurry Format is tested against predetermined specifications to ensure consistent product quality P2. be observed if the urine samples are stored at either 4 C or at 20 C DO NOT discard this precipitate and or spin down your samples to get rid of the turbidity this will significantly reduce your DNA yields Make sure to mix your samples thoroughly before processing 2 What If a variable speed centrifuge is not available e A fixed speed centrifuge can be used however reduced yields may be observed 3 What will happen if my centrifugation speed varied from the recommended speed e This may lead to the degradation of the genomic DNA or reduction in the total DNA yields 4 At what temperature should I centrifuge my samples e All centrifugation steps are performed at room temperature Centrifugation at 4 C will not adversely affect kit performance 5 What If added more or less of the specified reagents volume e Adding more or less from the specified volume may affect the quality and the quantity of the isolated DNA 6 What If I forgot to do a dry spin after my second wash e Your first DNA elution will be contaminated with the Wash Solution A This may dilute the DNA yield in your first elution and it may interfere with your downstream applications 7 Can I perform a third elution e Yes you can A third elution is possible but it is recommended that this elution is performed in a smaller volume 50 uL 8 Why do my samples show very low DNA yield e Some urine samples contain very little DNA This varies from individual t
3. Add 50 uL from 20mg mL Proteinase K to the precipitated slurry pellet Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes then proceed to Step 4 Option 3 Proceed directly to Step 4 if Option 1 or Option 2 is not required Add 500 uL Lysis Buffer A to the precipitated slurry pellet mix well by vortexing for 10 seconds Add 500 uL of 96 100 Ethanol to the mix from Step 4 mix well by vortexing for 10 seconds Place the 96 Well Filter Plate on top of a provided 96 Well Collection Plate Transfer 650 uL from the mixture from Step 5 including all resin into a well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 2 000 RPM for 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate and the 96 Well Collection Plate Note Ensure that all of the lysate from each well has passed through into the bottom plate If the entire lysate volume has not passed centrifuge for an additional 2 minutes Repeat Step 7 and Step 8 to transfer the rest of the mixture from Step 5 Apply 400 uL of Wash Solution A to each used well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 2 000 RPM for 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate and the bottom plate Note Ensure the entire Wash Solution A has passed through into the bottom plate by inspecting the 96 Well Filter Plate If the entire wash volume has not passed centrifuge for an additional 2
4. Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com gt k 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Urine DNA Isolation 96 Well Kit Slurry Format Product Insert Product 27100 Norgen s Urine DNA Isolation 96 Well Kit Slurry Format provides a fast reliable and simple procedure for high throughput isolation of DNA from 3 25 mL urine samples DNA found in urine can be divided into 2 basic categories The larger species genomic DNA is generally greater than 1 kb in size and appears to be derived mainly from cells shed into the urine The second species is smaller generally between 150 and 250 bp apoptotic DNA and derives at least in part from the circulation The second species is also considered as an RNA DNA hybrid as reported by Halicka et al 2000 Both types of DNA can be isolated reliably using this kit This kit is designed to process 96 samples of 3 25 mL of urine each Typical yields of DNA isolated will vary depending on the input sample with more concentrated samples tending to yield more DNA Norgen s Urine DNA Isolation 96 Well Kit Slurry Format can also be used for the isolation of viral DNA from urine Preparation time for a single sample is less than 30 minutes The purified urine DNA is compatible with PCR and Southern Blot analysis This kit is designed to process 96 samples of 3 25 mL urine each Kit Components
5. Vacuum 4 minutes rss y a a Wash three times with 400 s uL Wash Solution A Vacuum 3 minutes V aT p Pe i Dry by Vacuum 5min I Elute DNA with DNA ys H Elution Buffer B E1 100 pL 8 E2 100 pL Vacuum 3 min Purified Yotal DNA Rapid Flow Chart Procedure Using Centrifugation 3mL 25 mL urine Sample Add 0 3 mL Slurry B1 Solution A must be mixed well before every pipeting Vortex for 10 seconds SPIN 2 000 RPM 5 min Aaria Discard Supernatant E Add 0 5mL Lysis Buffer A 3 Vortex for 10 seconds I Add 0 5mL 96 100 Ethanol 3 Vortex for 10 seconds Ls y j Mix transfer 650uL into each well of ca the 96 Well Filter Plate i transfer twice Centrifuge 2 minutes at 2 000 RPM i Wash three times with 1 400 uL Wash Solution A Centrifuge 2 minutes at 3 000 RPM yi J y Dry by Centrifugation 5 minutes at 3 000 RPM 4 lt Ls eEution Buffer B E1 100 pL E2 100 uL CY Elute DNA with DNA Centrifuge 2 minutes at 3 000 RPM Purified Total DNA Frequently Asked Questions 1 If am not going to process my samples immediately how should I store my samples e We recommend the use of Norgen s Urine Preservative when collecting urine samples which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures Urine samples in the preservative should be stored at room temperature Turbidity or precipitation may
6. e can be used as the collection waste tray if desired Transfer 650 uL of the mixture from Step 5 including all resin into a well of the 96 Well Filter 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 4 minutes Turn off vacuum and ventilate the manifold Discard the flowthrough Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat Step 7 and Step 8 to transfer the rest of the mixture from Step 5 Apply 400 uL of Wash Solution A to each used well of the 96 Well Filter 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 3 minutes Turn off vacuum and ventilate the manifold Discard the flowthrough Repeat Step 10 to wash the used well a second time Apply 400 uL of Wash Solution A to each used well of the 96 Well Filter 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 3 minutes Turn off vacuum and ventilate the manifold Discard the flowthrough Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the vacuum manifold Apply vacuum for an additional 5 minutes in order to completely dry the plate Turn off vacuum and ventilate th
7. e in 2 convenient formats in a liquid format in Norgen s Urine Preservative Single Dose Ampules as well as in a dried format in Norgen s Urine Collection and Preservation Tubes Please see the Related Products table below e Do not spin down or filter the urine sample before proceeding with the isolation as this could decrease the DNA yield 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 Slurry B1 contains grey resin that will not disappear when warmed e Prepare a 400 mg mL stock solution approximately 1 7 x10 units mL of lysozyme as per supplier s instructions if bacterial gDNA isolation is needed e Preheat an incubator or heating block to 60 C e Prepare a working concentration of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution A This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Urine DNA isolation can be performed using either a vacuum manifold or centrifugation For purification using vacuum please follow the procedure outlined in Section A For purification using centrifugation please follow the procedure outlined in Section B e Urine inputs of 3 to 25 mL can be processed using either of the procedures ou
8. e manifold Replace the collection waste tray in the vacuum manifold with a provided 96 Well Elution Plate Complete the vacuum manifold assembly with the 96 Well Filter Plate Add 100 uL of Elution Buffer B to each used well of the plate Apply vacuum for 3 minutes Replace the used 96 Well Elution Plate in the vacuum manifold with the provided second 96 Well Elution Plate Complete the vacuum manifold assembly with the 96 Well Filter Plate Add 100 uL of Elution Buffer B to each used well of the plate Apply vacuum for 3 minutes Urine DNA is now ready for downstream applications Detailed Procedure Using Centrifugation Add 300 uL of Slurry B1 to each urine sample Mix well by vortexing for 10 seconds Note 1 Slurry B1 must be mixed well before every pipeting Note 2 The volume of Slurry B1 is fixed with all urine volumes ranging from 3 mL and up to 25 mL 10 11 12 13 14 15 Centrifuge for 2 minutes at 2 000 RPM then discard the supernatant carefully in order not to dislodge the precipitated slurry pellet Option 1 To Isolate bacterial gDNA with the human DNA e Add 12 uL of the previously prepared lysozyme to the precipitated slurry pellet Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes then proceed to Step 4 Option 2 Urine samples which contain large amounts of protein e Incase your urine sample contains large amount of proteins a Proteinase K treatment can be done as this step
9. ienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Urine DNA Isolation 96 Well Kit Slurry Format or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please 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 or call one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com References Halicka H D Bedner E and Darzynkiewicz Z 2000 Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis Exp Cell Res 260 248 256 M Abdalla and Y Haj Ahmad 2006 Urinary Proteomic and Genomic Profiles from Hepatitis C virus Hepatitis B Virus and Hepatocellular Carcinoma Patients Molecular amp Cellular Proteomics ASBMB S369 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 To
10. ll Free in North America 1 866 667 4362 2014 Norgen Biotek Corp P127100 7 M14
11. minutes Repeat Step 10 to wash the used wells a second time Apply 400 uL of Wash Solution A to each used well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 2 000 RPM for 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate and the bottom plate Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the bottom plate Centrifuge the assembly at maximum speed or 2 000 RPM for 5 minutes in order to completely dry the plate Stack the 96 Well Filter Plate on top of one of the provided 96 Well Elution Plates Add 100 uL of Elution Buffer B to each used well of the plate Centrifuge the assembly at maximum speed or 2 000 RPM for 2 minutes Replace the used 96 Well Elution Plate with the second provided 96 Well Elution Plate Add 100 uL of Elution Buffer B to each used well of the plate Centrifuge the assembly at maximum speed or 2 000 RPM for 2 minutes 7 Urine DNA is now ready for downstream applications Rapid Flow Chart Procedure Using Vacuum Manifold 3mL 25 mL urine Sample Add 0 3 mL Slurry B1 Solution A must be mixed well before every pipeting Vortex for 10 seconds SPIN 2 000 RPM 5 min Discard Supernatant ae ee Ce ee Add 0 5mL Lysis Buffer A Vortex for 10 seconds Add 0 5mL 96 100 Ethanol Vortex for 10 seconds 4 H Gad the 96 Well Filter Plate transfer twice Fee EE i Mix transfer 650uL into each well of
12. o individual based on numerous variables In order to increase the yield the amount of urine input could be increased 9 Why does my DNA does not perform well in downstream applications e lf a different Elution Buffer B was used other than the one provided in the kit the buffer should be checked for any components that may interfere with the application Common components that are known to interfere are high salts including EDTA detergents and other denaturants Check the compatibility of your Elution Buffer B with the intended use 10 What is the expected DNA yield from urine e The urinary DNA yield varies between individual samples Generally the DNA yield ranges between 50 ng 2 ug mL of urine sample Sometimes the DNA yields from urine are too low to be visualized on an agarose gel however the DNA yield is sufficient for most of the downstream applications including PCR and Southern hybridization Related Products Product Urine DNA Isolation Kit 18100 Urine DNA Isolation Kit for Exfoliated Cells or Bacteria 47050 Urine Collection and Preservation Tubes 50 cc 1 tube 50 tubes 18111 18113 Urine Collection and Preservation Tubes 15 cc 1 tube 50 tubes 18120 18122 Urine Collection and Preservation Tubes 5 cc 1 tube 50 tubes 18116 18118 Urine Preservative Single Dose 1 tube 50 tubes 18124 18126 Technical Assistance NORGEN s Technical Service Department is staffed by exper
13. roduct Use Limitations Norgen s Urine DNA Isolation 96 Well Kit Slurry Format is designed for research purposes only It is not intended for human or diagnostic use Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use Safety Information 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 I CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Lysis Buffer A 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 Procedure Notes prior to use e We recommend the use of Norgen s Urine Preservative when collecting urine samples which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures The components of the Urine Preservative allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA RNA or proteins Norgen s Urine Preservative is availabl
14. tlined below A Detailed Procedure Using Vacuum Manifold 1 Add 300 uL of Slurry B1 to each urine sample Mix well by vortexing for 10 seconds Note 1 Slurry B1 must be mixed well before every pipeting Note 2 The volume of Slurry B1 is fixed with all urine volumes ranging from 3 mL and up to 25 mL 2 Centrifuge for 2 minutes at 2 000 RPM then discard the supernatant carefully in order not to dislodge the precipitated slurry pellet 10 11 12 13 14 15 Option 1 To Isolate bacterial gDNA with the human DNA e Add 12 uL of the previously prepared lysozyme to the precipitated slurry pellet Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes then proceed to Step 4 Option 2 Urine samples which contain large amounts of protein e In case your urine sample contains large amount of proteins a Proteinase K treatment can be done at this step Add 50 uL of 20mg mL Proteinase K to the precipitated slurry pellet Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes then proceed to Step 4 Option 3 Proceed directly to Step 4 if Option 1 or Option 2 is not required Add 500 uL Lysis Buffer A to the precipitated slurry pellet mix well by vortexing for 10 seconds Add 500 uL of 96 100 Ethanol to the mix from Step 4 mix well by vortexing for 10 seconds Assemble the 96 Well Filter Plate and the vacuum manifold according to manufacturer s recommendations Note The provided 96 Well Collection Plat
Download Pdf Manuals
Related Search