Size-Select Kit for NGS Library Preparation
Contents
1. Ensure that no more than 10 ug of DNA is used as the input the input for each column The initial centrifugation Ensure that the initial 15 min centrifugation after and supernatant mixing the sample and the Size Select Additive and Clogged recovery was not Size Select Buffer was performed and the Column performed supernatant was transferred to a clean tube Centrifuge temperature too low Ensure that the centrifuge remains at room temperature throughout the procedure Temperatures below 15 C may cause precipitates to form that can cause the columns to clog DNA does not perform well in downstream applications DNA was not washed with the provided Wash Solution Traces of salt from the binding step may remain in the sample if the column is not washed for the specified wash times with the Wash Solutions 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 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 2013 Norgen Biotek Corp P1I53600 12. column If the entire volume has not passed spin for an additional minute 3 Column Wash a Apply 500 uL of Wash Solution I to the column and centrifuge for 1 minute at 8 000 x g 8 000 RPM Note Ensure the entire Wash Solution has passed through the column into the collection tube by inspecting the column If the entire 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 Il to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Note Ensure the entire Wash Solution II has passed through the column into the collection tube by inspecting the column If the entire volume has not passed spin for an additional minute d Discard the flowthrough and reassemble the spin column with its collection tube e Repeat steps 3c and 3d to wash the column another time with Wash Solution II Spin the column for 2 minutes at 14 000 x g 14 000 RPM in order to thoroughly dry the resin Discard the collection tube n 4 Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 30 uL of Elution Buffer to the column and let sit at room temperature for 1 minute c Centrifuge 1 minute at 14 000 x g 14 000 RPM Note 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 5 Storage of Pu
3. selection using Norgen s Size Select Kit for NGS Library Preparation Fragmented DNA Sample Add Size Select Additive Add Select Size Buffer Vortex Transfer supernatant to a clean tube Add Binding Solution Vortex E Bind to column SPIN J gt Wash once with Wash Solution Wash twice with Wash Solution Il SPIN 3 Dry spin Elute with Elution Solution SPIN Purified Sample 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 10 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 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 The Size Select Buffer can be used directly from the fridge at 4 C Ensure that all other solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution Il by adding 23 mL
4. D k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com Size Select Kit for NGS Library Preparation Product Insert Product 53600 Norgen s Size Select Kit for NGS Library Preparation provides a rapid simple and efficient procedure for the size selection and clean up of fragmented DNA The resulting DNA is suitable for use in next generation sequencing library preparation The kit selectively isolates DNA ranging in size from approximately 200 500 bp from a mixture of fragmented DNA and removes various impurities including un incorporated adaptors and enzymes without the use of magnetic beads or agarose gel extraction The kit is robust and is compatible with input volumes of up to 10 ug The purified and size selected DNA can then be used in subsequent PCR reactions for next generation sequencing library preparation Norgen s Size Selection and Purification Technology Size selection is based on first removing large DNA fragments from your mixture using the Size Select Buffer The small DNA remaining in the supernatant lt 400 500bp is then transferred to Norgen s spin column The DNA is preferentially purified from other impurities such as proteins and salts without the use of phenol chloroform or magnetic beads The process involves first mixing the fragmented and adaptor ligated DNA sample with the Size Select A
5. dditive and Size Select Buffer and centrifugation to remove DNA sizes gt 500 bp Next the Binding Solution is added to the supernatant and then is loaded onto a spin column Norgen s column binds nucleic acids in a manner that depends on ionic concentrations Thus only the nucleic acids will bind to the column while the contaminating proteins or nucleotides will be removed in the flowthrough The bound nucleic acid is then washed with the provided Wash Solutions in order to remove DNA sizes of lt 200bp such as smaller genomic DNA fragments unligated adaptors and adaptor dimers After washing the DNA is eluted in a 30 uL volume The kit is designed to process 25 samples Specifications Kit Specifications Column Binding Capacity 10 ug of DNA Maximum Column Loading Volume 600 uL Size of DNA Purified Approximately 200 400 bp Time to Complete 10 Purifications 25 minutes Minimum Elution Volume 30 uL Advantages e Column purification DNA is column cleaned eliminating hazardous and labor intensive phenol based procedures or costly magnetic beads e Selection of DNA sizes of 200bp 500bp for NGS Efficient size separation and removal of buffers and enzymes in addition to unincorporated adaptors e Rapid procedure Size select and concentrate samples in 25 minutes e Provides high quality DNA fragments The purified DNA fragments are of the highest quality and can be used for next generation sequencing
6. library preparation Kit Components Component Contents Size Select Additive 0 3 mL Size Select Buffer 2x1mL Binding Solution 12 mL Wash Solution 15 mL Wash Solution II 6 mL Elution Buffer 2mL Spin Columns 25 Collection Tubes 25 Elution tubes 1 7 mL 25 Product Insert 1 Storage Conditions and Product Stability The Size Select Buffer should be kept tightly sealed and stored upright away from light at 4 C All other solutions should be kept tightly sealed and stored at room temperature 15 25 C for up to 1 year without showing any reduction in performance 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 The Binding Solution and Wash Solution 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 Customer Supplied Reagents and Equipment e Benchtop microcentrifuge e Micropipettors e Nuclease free water e 96 100 ethanol to be added to wash buffer Flow Chart Procedure for DNA size
7. of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 29 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e The maximum sample input volume that can be processed is 190 uL 1 Size Select Preparation a The protocol can be used to process up to 190 uL of fragmented DNA If the volume is less than 190 uL add nuclease free water to adjust the volume of the sample to 190 uL b Add 10 uL of Size Select Additive and mix c Add 71 uL of Size Select Buffer and mix thoroughly by vortexing for 10 seconds d Centrifuge at 10 000 x g for 15 minutes Ensure the back of the microcentrifuge tube is oriented to the outside of the rotor A pellet may not be visible but this will ensure its location will be on the back wall of the tube e Carefully transfer the supernatant to a clean microcentrifuge tube not provided Do not disturb the DNA pellet which will be present on the back wall of the microcentrifuge tube 2 Sample Binding a Add 400 uL of Binding Solution and mix by vortexing f Assemble a spin column with one of the provided collection tubes g Apply the mixture onto the column and centrifuge for 1 minute at 8 000 RPM h Discard the flowthrough and reassemble the column with its collection tube Note Ensure the entire solution has passed through the column into the collection tube by inspecting the
8. rified and Size selected DNA The purified DNA samples should be stored at 20 C or can be used immediately in subsequent steps of a library preparation protocol Related Products Product Total RNA Purification Kit 17200 PCR Purification Kit 14400 10X RNA Fragmentation Buffer 53500 MiniSizer 50 bp DNA Ladder 11200 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 Do not exceed the recommended amounts of Colman has bet ne starting materials The amount of starting material slog ed may need to be decreased if the column shows 99 clogging below the recommended levels See also Clogged Column below An alternative elution It is recommended that the Elution Buffer supplied Poor DNA solution was used with this kit be used for maximum DNA recovery Recovery aa O ni gs d Ensure that the appropriate amount of the Size Select Additive and Size Select Buffer is added to Size Select Buffer were the sample not used Ethanolwas not added Ensure that the correct volume of 96 100 ethanol is added to the supplied Wash Solution II prior to to Wash Solution II tise High amounts of DNA in
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