27600 - Protocol (50 prep)
Contents
1. column SPIN 3 Wash once with Buffer SK Wash twice with Wash Solution A SPIN J Elute DNA with Elution Solution B SPIN J Purified Total DNA Procedures 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 g force Notes Prior to Use e All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature 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 Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of Wash Solution A by adding 42 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 60 mL The label on the bottle h2. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com Stool DNA Isolation Kit Product Insert Product 27600 Norgen s Stool DNA Isolation Kit provides a convenient and rapid method to isolate total DNA from fresh or frozen stool samples The kit can also be used to isolate DNA from stool samples preserved using Norgen s Stool Nucleic Acid Collection and Transport Tubes The universal protocol conveniently allows for the isolation of total genomic DNA from all the various microorganisms and host cells found in the stool sample simultaneously The kit removes all traces of humic acid using the provided Bead Tubes and a combination of chemical and physical homogenization and lysis A simple and rapid spin column procedure is then used to further purify the DNA The purified DNA is of the highest quality and is fully compatible with downstream PCR applications as all humic acid substances and PCR inhibitors are removed during the isolation Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The process involves first adding the stool sample and Lysis Buffer L to a provided Bead Tube and vortexing briefly to mix Lysis Additive A is then added to the Bead Tube and the tube is vortexed in order to efficiently and rapidly homoge
3. well in downstream PCR reaction Take steps to optimize the PCR conditions being used applications including varying the amount of template changing the conditions need to be optimized source of Taq polymerase looking into the primer design and adjusting the annealing conditions Related Products Product Stool Nucleic Acid Collection and Transport Tubes 50 Tubes 45650 Stool Nucleic Acid Isolation Kit 45600 Stool Total RNA Purification Kit 49500 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 ort norgenbiotek com email at techsu 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 P127600 6 M14
4. as a box that may be checked to indicate that the ethanol has been added 1 Lysate Preparation a Add up to 200 mg of stool sample to a provided Bead Tube and add 1 mL of Lysis Buffer L Vortex briefly to mix stool and Lysis buffer L For stool samples that have been preserved using Norgen s Stool Nucleic Acid Collection and Transport Tubes Cat 45650 add 200 uL of preserved sample to a provided Bead Tube and add 800 uL of Lysis Buffer L Vortex briefly to mix stool and Lysis Solution b Add 100 uL of Lysis Additive A and vortex briefly c Secure tube horizontally on a flat bed vortex pad with tape or secure the tube in any commercially available bead beater equipment e g Scientific Industries Disruptor Genie Vortex for 3 minute at maximum speed d Centrifuge the tube for 2 minute at 14000 x g 14 000 RPM e Transfer up to 600 uL of supernatant to a DNAase free microcentrifuge tube not provided f Add 100 uL of Binding Buffer I mix by inverting the tube a few times and incubate for 10 minutes on ice Spin the lysate for 2 minutes to pellet any cell debris Using a pipette transfer up to 700 uL of supernatant avoid contacting the pellet with the pipette tip into a 2 mL DNAase free microcentrifuge tube not provided i Add an equal volume of 70 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 ze 2 B
5. cations 30 minutes Universal method to detect microorganisms and host cell simultaneously in stool samples Advantages e e Rapid and convenient rapid spin column format e Remove all humic acid from DNA samples e Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents Isolate high quality total DNA for down steam applications should remain stable for at least 1 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only Itis 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 Customer Supplied Reagents and Equipment You must have the following in order to use the Stool DNA Isolation Kit Benchtop microcenrifuge DNAse free microcentrifuge tubes 96 100 ethanol 70 ethanol Flat bed vortex or bead beater equipment Flow Chart Procedure for Purifying Total DNA using Norgen s Stool DNA Isolation Kit Add stool sample Lysis Buffer L and Lysis Additive A to Bead Tube Vortex for 3 minutes Centrifuge Transfer lysate Add Binding Buffer I Incubate for 10 minutes on ice a Add Ethanol SPIN J Transfer lysate Bind to
6. dded to the lysate lysate before binding to the column See eee Ensure that 42 mL of 96 100 ethanol is added to Solution A the supplied Wash Solution A prior to use olution Ensure that the Lysis Additive A is added Also ensure Binding Solution is added to the lysate and that it is Eluted DNA incubated on ice for 10 minutes prior to spinning down sample is brown the lysate Avoid any contact with the pellet or surface residue when collecting the supernatant after the 5 minute spin during Sample Preparation DNA does coe ie Ensure that the provided Lysis Additive A is added to the lysate not perform the lysate well in downstream Seat ee applications DNA was not Traces of salt from the binding step may remain in the washed with the provided Buffer SK and Wash Solution A sample if the column is not washed three times with the provided Buffer SK and Wash Solution A Salt may interfere with downstream applications and thus must be washed from the column Ethanol carryover 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 Problem Possible Cause Solution and Explanation Binding Buffer Ensure that the Binding Buffer is added to the lysate DNA does was not added to and that it is incubated on ice for 10 minutes prior to the lysate spinning down the lysate not perform
7. inding to Column a Assemble a spin column with one of the provided collection tubes b Apply 600 uL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 2 3 500 x g 6 000 RPM Discard the flowthrough and reassemble the spin column with the collection tube Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute at 14 000 x g 14 000 RPM c Repeat step 2b with the remaining volume of lysate mixture 3 Column Wash a Apply 500 uL of Buffer SK to the column and centrifuge for 1 minute Note Ensure the entire Buffer SK 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 Discard the flowthrough and reassemble the spin column with its collection tube c Apply 500 uL of Wash Solution A to the column and centrifuge for 1 minute d Discard the flowthrough and reassemble the spin column with its collection tube e Repeat 3c and 3d f Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 DNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Buffer B to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a 1 minute spin at 14 000 x g 14 000 RPM N
8. nize the sample extract the DNA and remove all humic acids The sample is then centrifuged and the supernatant is transferred to a DNAse free microcentrifuge tube Binding Buffer is added and the lysate is incubated for 10 minutes on ice The lysate is then spun for 2 minutes to pellet any cell debris the supernatant is collected an equal volume of 70 ethanol is added to the lysate and the solution is loaded onto a spin column Norgen s resin binds nucleic acids in a manner that depends on ionic concentrations thus only the DNA will bind to the column while the proteins are removed in the flowthrough or retained on top of the resin The bound DNA is then washed using the provided Buffer SK and Wash Solution A and the purified DNA is eluted using the Elution Buffer B The purified total DNA is free of all inhibitors including humic acid and can be used in sensitive downstream applications including PCR Kit Components Component Product 27600 50 preps Lysis Buffer L 60 mL Lysis Additive A 6 mL Binding Buffer 7 mL Buffer SK 30 mL Wash Solution A 18 mL Elution Buffer B 8 mL Bead Tube 50 Mini Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Specifications Kit Specifications Maximum Stool Input 200 mg fresh or frozen stool Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL Time to Complete 10 Purifi
9. ote the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute d Optional An additional elution may be performed if desired by repeating steps 4b and 4c using 50 pL of Elution Buffer The total yield can be improved by an additional 20 30 when this second elution is performed 5 Storage of DNA The purified genomic DNA can be stored at 2 8 C for a few days For longer term storage 20 C is recommended Troubleshooting Guide Problem Possible Cause Solution and Explanation Depending on the type of stool further vortexing with the flat bed vortex or bead beater equipment may be Homogenization required However it is not recommended to increase was incomplete the vortex time to longer than 5 minutes at maximum speed Also ensure that the maximum input of 200 mg of stool is not exceeded as this may also cause incomplete homogenization a a It is recommended that the Elution Buffer B supplied Used with this kit be used for maximum DNA recovery Ensure that the provided Lysis Additive A is added to Poor DNA Lysis Additive A separate humic acid and increase DNA yield Also an Recovery was not added to incubation can be preformed at 65 C for 10 minutes the lysate after addition of the Lysis Additive A and prior to vortexing to maximize DNA recovery Ethanol was not Ensure that an equal amount of ethanol is added to the a
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