ChargeSwitch® gDNA Plant Kit
Contents
1. 25 26 27 28 Remove the plate from the 96 well Magnetic Separator 16 Add 1 ml ChargeSwitch Wash Buffer W12 to the samples Shake at medium speed e g pulse 10 seconds to evenly distribute the magnetic beads within the solution or mix by gently pipetting up and down 5 times Move the plate to the 96 well Magnetic Separator Wait for 60 90 seconds Slowly aspirate all of the supernatant and discard leaving behind the pellet of beads Remove plate from the 96 well Magnetic Separator Add 150 ul of ChargeSwitch Elution Buffer E6 Note You may vary the elution volume 50 100 ul to suit the application Do not use volumes lt 50 ul for eluting DNA Shake the plate rapidly for 1 minute or mix by pipetting up and down until ChargeSwitch Magnetic Beads are completely dispersed and no clumps are visible Incubate at room temperature for 1 minute Note If lt 150 ul ChargeSwitch Elution Buffer is used for elution increase the incubation time to 5 minutes Move plate to the 96 well Magnetic Separator Wait for 1 minute Slowly aspirate and transfer supernatant containing the DNA to a 96 x 300 ul U bottom microplate without disturbing the pellet Discard the used ChargeSwitch Magnetic Beads Do not reuse the ChargeSwitch Magnetic Beads For DNA quantitation refer to next page Storing DNA Store the purified gDNA at 20 C or use immediately for the desired downstream application Avoid2. For individual lot test results and more information visit www invitrogen com to download the Certificate of Analysis Accessory Products Additional Products The table below lists additional products available from Invitrogen that may be used with the ChargeSwitch gDNA Plant Kit In addition the table lists a selection of ChargeSwitch gDNA Kits that are available for purification of genomic DNA from other sources For more information about these and other ChargeSwitch gDNA Kits refer to our website at www invitrogen com or call Technical Support see page 23 Product Amount Catalog no MagnaRack Magnetic Rack 1 rack CS15000 96 well Magnetic Separator 1 rack CS15096 96 Deep Well Block 2 2 ml 1 case of 50 CS15196 ChargeSwitch gDNA Micro Tissue Kit 50 purifications CS11203 ChargeSwitch gDNA Mini Tissue Kit 25 purifications CS11204 ChargeSwitch gDNA 10 20 ul Blood Kit 96 purifications CS11010 ChargeSwitch gDNA 50 100 ul Blood 50 purifications CS11000 Kit ChargeSwitch gDNA 1 ml Blood Kit 20 purifications CS11001 ChargeSwitch gDNA 1 ml Serum Kit 50x 1 ml purifications CS11040 ChargeSwitch gDNA Mini Bacteria Kit 50 purifications CS11301 ChargeSwitch gDNA Normalized 50 purifications CS11020 Buccal Cell Kit ChargeSwitch gDNA Buccal Cell Kit 50 purifications CS11021 Quant iT DNA Assay Kit High 1000 assays Q33120 Sensitivity Quant iT DNA Assay Kit Broad Range 1000 a
3. Maintain a sterile environment when handling DNA to avoid any contamination from DNases Ensure that no DNases are introduced into the solutions supplied with the kit Make sure all equipment that comes in contact with DNA is sterile including pipette tips and tubes When removing supernatant aspirate slowly to ensure that the pellet of beads is not disturbed Make sure that the ChargeSwitch Wash Buffer is removed completely before elution and the ChargeSwitch Magnetic Beads are fully resuspended during the elution step Follow these safety guidelines when using the ChargeSwitch Kits Treat all reagents supplied in the kit as potential irritants Always wear a suitable lab coat disposable gloves and protective goggles If a spill of the buffers occurs clean with a suitable laboratory detergent and water If the liquid spill contains potentially infectious agents clean the affected area first with laboratory detergent and water then with 1 v v sodium hypochlorite or a suitable laboratory disinfectant Continued on next page General Information Continued Handling Magnetic Beads Follow the recommendations below for best results Do not freeze ChargeSwitch Magnetic Beads as freezing damages the property of the beads for nucleic acid purifications Store the beads at room temperature Always keep the ChargeSwitch Magnetic Beads in solution Do not allow the beads to dry including durin
4. Starting Material Up to 100 mg plant sample Bead Binding Capacity 1 mg bead binds 5 10 ug gDNA Bead Size 1 um Bead Concentration 25 mg ml Bead Storage Buffer 10 mM MES pH 5 0 10 mM NaCl 0 1 Tween 20 Elution Volume 150 ul DNA Yield Up to 7 ug Use of the ChargeSwitch gDNA Plant Kit catalog no CS18000 10 has been demonstrated on the Tecan Genesis robotic workstation to purify gDNA in a fully automated system from large numbers of plant samples in a 96 well format Other liquid handling robots are suitable provided that each robot is equipped with a gripper arm a 96 well magnetic separator and other additional hardware as described on page 13 This manual provides general guidelines and a protocol that may be used to develop a script for your robot For more information see www invitrogen com or call Technical Support page 23 Experimental Overview Experimental The figure below illustrates the basic steps necessary to Outline purify genomic DNA using the ChargeSwitch gDNA Plant Kits Lyse sample Bead charge Bind DNA to ChargeSwitch SS on magnetic beads p lt 60 Charge on Wash beads containing DNA pH 7 0 to remove contaminants Charge off pH 8 5 Elute DNA from beads General Information Safety Information To maximize DNA yield follow these important recommendations when processing your samples Resuspend the ChargeSwitch Magnetic Beads thoroughly before use
5. Purification Procedure Automated Protocol page 13 Purification Procedure Manual Protocol Introduction MagnaRack This procedure is designed for purifying genomic DNA from plant samples using microcentrifuge tubes If you are using an automated liquid handling system to process the samples proceed with the Purification Procedure Automated Protocol page 13 Use this Manual Protocol with the ChargeSwitch gDNA Plant Kit catalog no CS18000 The MagnaRack Magnetic Rack available from Invitrogen catalog no CS15000 is a two piece magnetic separation rack for use in protocols with magnetic beads The MagnaRack Magnetic Rack consists of a magnetic base station and a removable tube rack The tube rack can hold up to 24 microcentrifuge tubes The tube rack fits onto the magnetic base station in two different positions associating the row of 12 neodymium magnets with a single row of 12 tubes for simple on the magnet and off the magnet sample processing see figure below For more information visit www invitrogen com or call Technical Support page 23 ie Continued on next page Purification Procedure Manual Protocol Continued Materials You will need the following items Needed Plant sample lysate see Sample Preparation page 6 MagnaRack previous page 1 5 ml microcentrifuge tubes Adjustable pipettes and aerosol barrier pipette tips Components supplied with th
6. Wash Buffer W12 5 Waste 6 96 Well Magnetic Separator 7 Shaker 8 96 well sample tray 9 ChargeSwitch Elution Buffer E6 10 96 well U bottom microtiter plate for final elution 16 E Well Magnetic Separator Continued on next page Purification Procedure Automated Protocol Continued O O QE END Pa Now To maximize DNA yield using the automated protocol follow these important recommendations below when processing samples e Ensure that the robotic tips enter the wells of the plates without interfering with the pellet of beads e When removing supernatant leave samples on the 96 Well Magnetic Separator and aspirate slowly to ensure that the pellet of beads is not disturbed e When resuspending pelleted ChargeSwitch Magnetic Beads ensure that all beads are fully resuspended to maximize DNA recovery e To maximize DNA yield make sure that all Wash Buffer is removed from the beads before adding the Elution Buffer and the beads are fully resuspended during the elution step Continued on next page 17 Purification Procedure Automated Protocol Continued Automated Follow the protocol below to purify genomic DNA from plant Protocol samples The volumes given are on a per sample basis The protocol below describes the use of 96 well deep well plates page 15 and can also be used with microcentrifuge tubes Start with plant lysates prepared as descr
7. add 1 ml ChargeSwitch Lysis Buffer L18 to the tissue from Step 2 or for samples rich in polysaccharides and polyphenolics add 1 ml ChargeSwitch Lysis Buffer L18 containing Reagent A see previous page for recipe to the tissue from Step 2 Note If the solution is very viscous add more Lysis Buffer L18 Also see Step 6 below Add 2 ul RNase A to the samples 5 Prepare lysate by homogenizing the pieces of soft tissue with a tissue homogenizer or grinder or by vortexing the ground tissue lyophilized sample until sample is completely resuspended 6 Add 100 ul 10 SDS to 1 ml plant lysate If you varied the amount of ChargeSwitch Lysis Buffer L18 in Step 3 ensure to maintain a ratio of 10 1 for ChargeSwitch Lysis Buffer L18 to SDS Incubate at room temperature for 5 minutes 8 Add 400 ul ChargeSwitch Precipitation Buffer N5 to the lysate Mix by inversion or vortexing for 10 seconds until a precipitate forms If different amounts of ml ChargeSwitch Lysis Buffer L18 are used maintain a ratio of 10 4 for ChargeSwitch Lysis Buffer L18 to ChargeSwitch Precipitation Buffer N5 9 Centrifuge at maximum speed for 5 minutes at room temperature to produce a clear lysate 10 Transfer the clear lysate to a new sterile 1 5 ml microcentrifuge tube for manual purification or a 96 x 2 ml deep well plate page 15 for automated purification 11 Proceed to Purification Procedure Manual Protocol next page or
8. charge dependent on the pH of the surrounding buffer to facilitate nucleic acid purification In low pH conditions the ChargeSwitch Magnetic Beads have a positive charge that binds the negatively charged nucleic acid backbone see figure below Proteins and other contaminants are not bound and are simply washed away in an aqueous wash buffer To elute nucleic acids the charge on the surface of the ChargeSwitch Magnetic Beads is neutralized by raising the pH to gt 8 5 using a low salt elution buffer see figure below Purified DNA elutes instantly into this elution buffer and is ready for use in downstream applications ot ed Low pH High pH Continued on next page Overview Continued Advantages System Specifications Automation Use of the ChargeSwitch gDNA Plant Kit to isolate genomic DNA provides the following advantages e Uses magnetic bead based technology to purify genomic DNA without the need for hazardous chemicals centrifugation or vacuum manifolds e Rapid and efficient purification of genomic DNA from various plant samples in less than 15 minutes following sample preparation e Minimal contamination with RNA e Allows automated processing of large numbers of samples catalog no CS18000 10 in 96 well plates using a liquid handling robot e Purified genomic DNA demonstrates improved performance in downstream applications including PCR restriction enzyme digestion and Southern blotting
9. corn cotton prepare the lysate using Reagent A as described below and on the next page e Plant samples up to 100 mg e 1 5 ml microcentrifuge tubes and a microcentrifuge e 96x2ml deep well plate page 13 automated protocol e Tissue homogenizer grinder or mortar and pestle depending on the sample e Adjustable pipettes and aerosol barrier pipette tips e Optional Reagent A see below for recipe Components supplied with the kit e ChargeSwitch Lysis Buffer L18 e RNase A e ChargeSwitch Precipitation Buffer N5 e 10 SDS 1 Prepare 10 ml Reagent A 300 mM CaCh 15 polyvinylpyrolidone fresh as follows for plant samples rich in polysaccharides and polyphenolics CaCl 0 441 g Polyvinylpyrolidone 10 000 average MW 15g ChargeSwitch Lysis Buffer L18 10 ml 2 Mix well 3 To 900 pl ChargeSwitch Lysis Buffer L18 add 100 pl Reagent A and use this buffer to prepare samples see next page Continued on next page Sample Preparation Continued Preparing Plant Lysate Procedure to prepare lysate from up to 100 mg plant tissue 1 Chill the Precipitation Buffer N5 on ice 2 For hard plant tissue freeze the tissue in liquid nitrogen and grind frozen tissue to powder using mortar and pestle Let liquid nitrogen evaporate before proceeding to Step 3 For soft non fibrous plant tissue cut the tissue into small pieces For lyophilized samples proceed to Step 3 3 At room temperature
10. in solution at all times and are not dried Dried beads are non functional Eluate containing DNA is discolored Magnetic pellet disturbed during elution e Place the sample on the MagnaRack or on the 96 well Magnetic Separator until the beads form a tight pellet e Remove the eluate to a sterile microcentrifuge tube or sterile microtiter plate without disturbing the bead pellet DNA is sheared or degraded Lysate mixed too vigorously e Use appropriate pipette tip set to a volume lower than the total volume of the solution to mix the sample e Pipet up and down gently to mix Bubbles formed during mixing steps Make sure that the pipette tip is submerged in the solution during mixing DNA repeatedly frozen and thawed Aliquot DNA and store at 4 C or 20 C Avoid repeated freezing and thawing DNA contaminated with DNases Maintain a sterile environment while working e g wear gloves and use DNase free reagents 22 Technical Support Contact Us MSDS Requests Limited Warranty For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters European Headquarters Invitrogen Corporation Invitrogen Ltd 1600 Faraday Avenue Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel T
11. repeated freezing and thawing of gDNA 19 DNA Quantitation DNA Yield 20 Perform DNA quantitation using UV absorbance at 260 nm or Quant iT Kits UV Absorbance 1 Prepare a dilution of the DNA solution in 10 mM Tris HCl pH 7 5 Mix well Measure the absorbance at 260 nm A260 of the dilution in a spectrophotometer using a cuvette with an optical path length of 1 cm blanked against 10 mM Tris HCl pH 7 5 2 Calculate the concentration of DNA using the formula DNA ug ml A200 x 50 x dilution factor For DNA A2 1 for a 50 ug ml solution measured in a cuvette with an optical path length of 1 cm Quant iT Kits The Quant iI Kits see page vi for ordering information provide a rapid sensitive and specific fluorescent method for dsDNA quantitation The kit contains a state of the art quantitation reagent DNA standards for standard curve and a pre made buffer to allow fluorescent DNA quantitation using standard fluorescent microtiter plate readers fluorometers or the Qubit Quantitation Fluorometer available from Invitrogen Troubleshooting Refer to the table below to troubleshoot problems that you may encounter when purifying genomic DNA with the kit Introduction Problem Cause Solution Low DNA yield Incomplete lysis Reduce the amount of starting material Be sure to add SDS during lysis Poor quality of starting material Use fresh sample and proc
12. S18000 10 to process large numbers of samples in 96 well format using an automated liquid handling robot and magnetic separator If you wish to process small numbers of samples individually see Purification Procedure Manual Protocol page 8 The ChargeSwitch chemistry is ideal for purification of DNA using liquid handling robots by avoiding the need for centrifugation steps vacuum manifolds or the use of ethanol or chaotropic salts You will need the following hardware to perform automated processing of plant samples using the ChargeSwitch gDNA Plant Kit e Any liquid handling robotic workstation with a gripper arm e Appropriate tips for liquid dispensing and aspiration e 96 well magnetic separator see next page e Plate shaker capable of 1500 1800 rpm optional for use with deep well plates if desired Continued on next page 13 Purification Procedure Automated Protocol Continued 96 Well Magnetic Separator The 96 well Magnetic Separator available from Invitrogen catalog no CS15096 is a magnetic separation rack suitable for use in protocols with magnetic beads The rack can hold up to 96 samples in a deep well plate The deep well plate fits onto the magnetic separator associating the array of 24 neodymium magnets with the samples for simple processing of magnetic samples see figures below For more information see www invitrogen com or contact Technical Support page 23 Tip Sel
13. e kit ChargeSwitch Magnetic Beads ChargeSwitch 10 Detergent D1 ChargeSwitch Wash Buffer W12 ChargeSwitch Elution Buffer E6 Continued on next page Purification Procedure Manual Protocol Continued Binding DNA 10 Follow the protocol below to bind genomic DNA to ChargeSwitch Magnetic Beads The volumes given are on a per sample basis Start with lysed plant samples page 6 1 Thoroughly vortex the tube containing the ChargeSwitch Magnetic Beads to fully resuspend the beads in the storage buffer Add 100 ul ChargeSwitch 10 Detergent D1 to the 1 2 ml lysate from Step 10 page 7 Add 40 ul resuspended ChargeSwitch Magnetic Beads Mix gently by pipetting up and down 5 times using a 1 ml pipette tip set to 900 ul without forming any bubbles to evenly distribute the ChargeSwitch Magnetic Beads within the solution Important Avoid forming bubbles by ensuring that the pipette tip is submerged during mixing and by pipetting up and down gently Incubate at room temperature for 1 minute Place tubes on the MagnaRack on the magnet position until the ChargeSwitch have formed a tight pellet Without removing the tubes from the magnet carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet see figure on page 5 Proceed immediately to Washing DNA next page Cont
14. earch use only Not intended for any animal or human therapeutic or diagnostic use Tecan Genesis is a registered trademark of Tecan Group AG invitrogen Corporate Headquarters Invi Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country ific contact information visit our web site at www invitrogen com
15. ection 14 You may use any tips of choice to dispense and aspirate liquid during the purification procedure Consider the following factors when choosing an appropriate tip to use e Fixed vs disposable tips e Tip size vs head size e Conductive or non conductive e Sterile or non sterile e Filtered or non filtered Continued on next page Purification Procedure Automated Protocol Continued Materials You will need the following materials Needed 2 Plant sample lysates see Sample Preparation for Manual and Automated Protocol page 6 Liquid handling robot configured to process samples in 96 well plates 96 well Magnetic Separator see previous page 96 x 2 ml deep well plate page vi 96 x 300 ul U bottom microplate Greiner catalog no 650201 Components supplied with the kit ChargeSwitch Magnetic Beads ChargeSwitch 10 Detergent D1 ChargeSwitch Wash Buffer W12 ChargeSwitch Elution Buffer E6 Continued on next page 15 Purification Procedure Automated Protocol Continued Deck Set Up Once you have the required hardware you will need to configure the deck of your liquid handling robot appropriately to process samples You may use any suitable configuration of your choice An example is provided below Location Trough Contents Plate 1 96 well sample tray start position 2 ChargeSwitch Detergent D1 3 ChargeSwitch Magnetic Beads 4 ChargeSwitch
16. ents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 23 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology 24 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and no
17. ess immediately after collection or freeze the sample at 80 C or in liquid nitrogen The yield and quality of DNA isolated depends on the type and age of the starting material Incorrect handling of ChargeSwitch Magnetic Beads Vortex the tube containing the ChargeSwitch Magnetic Beads to fully resuspend the beads before adding them to your sample Pellet of beads disturbed or lost during binding or washing steps e Keep the sample on the MagnaRack or on the 96 well Magnetic Separator when removing supernatant during the binding or washing steps e Remove the supernatant without disturbing the pellet of beads Incorrect elution conditions e After adding ChargeSwitch Elution Buffer E6 to the sample pipet up and down to resuspend the ChargeSwitch Magnetic Beads before incubation e Do not use water to elute DNA Use ChargeSwitch Elution Buffer E6 Continued on next page 21 Troubleshooting Continued Problem Cause Solution No DNA recovered Water used for elution Do not use water for elution The elution buffer must have a PH 8 5 9 0 or the DNA will remain bound to the ChargeSwitch Magnetic Beads Use ChargeSwitch Elution Buffer E6 ChargeSwitch Magnetic Beads stored or handled improperly e Store beads at room temperature Do not freeze the beads as they will become irreparably damaged e Make sure that the beads are
18. g washing procedures as this renders the beads non functional Before using the ChargeSwitch Magnetic Beads resuspend thoroughly in the storage buffer by vortexing before removal from the storage tube During mixing steps avoid forming bubbles by using an adjustable pipette set to a specific volume as directed in the protocol and by pipetting up and down gently with the pipet tip submerged in the solution To aspirate the supernatant after bead washing place the pipette tip away from the beads by angling the pipette such that the tip is pointed away from the pellet and carefully remove the supernatant without disturbing or removing any beads see figure below lt Pipette tip lt Magnetic pellet Discard ChargeSwitch Magnetic Beads after use Do not reuse the beads Sample Preparation Introduction Plant Samples Materials Needed Preparing Reagent A Instructions for isolating genomic DNA gDNA from plant tissue are described below Read the entire instructions before starting the purification procedure The ChargeSwitch gDNA Plant Kit is suitable for isolating plant gDNA from a large variety of plant samples including leaves tissues sprouts shoots and seeds To obtain high yield of DNA and minimize DNA degradation use young plant samples such as leaves and freeze sample in liquid nitrogen immediately after collection For samples rich in polysaccharides and polyphenolics such as pine
19. ibed on page 6 1 Vortex the tube containing the ChargeSwitch Magnetic Beads to fully resuspend and evenly distribute the beads in the storage buffer 2 Add 100 ul 10 ChargeSwitch Detergent D1 to the 1 2 ml lysate from Step 10 page 7 Add 40 ul resuspended ChargeSwitch Magnetic Beads Shake at medium fast speed e g pulse 10 seconds to evenly distribute the ChargeSwitch Magnetic Beads within the solution You may also mix by gently pipetting up and down 5 times using a 1 ml pipette tip set to 900 ul without forming any bubbles Incubate at room temperature for 1 minute Move the plate to the 96 well Magnetic Separator Wait for 60 90 seconds Slowly aspirate all of the supernatant and discard leaving behind the pellet of beads oo ECON SOT 9 Remove the plate from the 96 well Magnetic Separator 10 Add 1 ml ChargeSwitch Wash Buffer W12 to the samples 11 Shake at medium speed e g pulse 10 seconds to evenly distribute the magnetic beads within the solution or mix by gently pipetting up and down 5 times 12 Move the plate to the 96 well Magnetic Separator 13 Wait for 60 90 seconds 14 Slowly aspirate all of the supernatant and discard leaving behind the pellet of beads Continued on next page 18 Purification Procedure Automated Protocol Continued Automated Protocol continued from previous page Protocol 15 continued 17 18 19 20 21 22 23 24
20. inued on next page Purification Procedure Manual Protocol Continued Washing DNA Remove tubes from the magnet 2 Add 1ml ChargeSwitch Wash Buffer W12 to the tube and gently pipet up and down 5 times to resuspend the beads using a 1 ml adjustable pipette set to 900 ul to mix without forming bubbles 3 Place tubes on the magnet until the beads have formed a tight pellet and the supernatant is clear 4 Without removing the tubes from the magnet carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet Remove tubes from the magnet Add 1 ml ChargeSwitch Wash Buffer W12 to the tube and gently pipet up and down 5 times to resuspend the beads using a 1 ml adjustable pipette set to 900 pl to mix without forming bubbles 7 Place tubes on the magnet until the beads have formed a tight pellet and the supernatant is clear 8 Without removing the tubes from the magnet carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet 9 Proceed immediately to Eluting DNA next page Continued on next page 11 Purification Procedure Manual Protocol Continued Eluting DNA 1 Remove the tube containing the pelleted magnetic beads from the magnet There should be no supernatant in the tube Add 150 u
21. invitrogen ChargeSwitch gDNA Plant Kit For purification of genomic DNA gDNA from plant samples Catalog nos CS18000 and CS18000 10 Version C 13 November 2006 25 0827 Table of Contents Table of Coments sa DR ES SS Ee ili Kit Contents and Storage siese ee ede Se ese Se edge De iv Product Qualification ccc eee eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee v Accessory Products eee vi OVYELVIEW daa os De Sarid Dd Dd DE od Bnd nh Be eh ink ome 1 Experimental Overview cceeeeececeeeessseeeeeeeeeeeeees 3 General Information ee ee 4 Sample Preparation ccccccccccceeeeeseeeeeeeeeeeeeeeeeeeees 6 Purification Procedure Manual Protocol 0008 8 Purification Procedure Automated Protocol 13 DNA uantitaioss se tas tates tata cat teases tae cats 20 TroubleshootiNg ss EE DE EE EE Ee EE EE Eg Ee 21 Technical SUPPOrE ie Es Ee deed eo dd gs 23 Purchaser Notification 2 c0s c 0 c eccedseeeeeeeeceneeeeeceeeceees 24 Kit Contents and Storage Types of Kits Shipping and Storage Kit Contents This manual is supplied with the following products Product Catalog no Purification Protocol ChargeSwitch gDNA Plant CS18000 Manual Kit CS18000 10 Automated All components of the ChargeSwitch gDNA Plant Kit are shipped at room temperature Upon receipt store components as follows e Store RNase A a
22. l of ChargeSwitch Elution Buffer E6 Note You may vary the elution volume 50 100 ul to suit the application Do not use volumes lt 50 ul for eluting DNA Pipet up and down gently 15 30 times using an adjustable pipette set to 100 pl adjust to any variations on the ChargeSwitch Elution Buffer volume used until ChargeSwitch Magnetic Beads are completely dispersed and no clumps are visible Note If a few grainy clumps remain proceed to the next step This will not adversely affect the result Incubate at room temperature for 1 minute Place tubes on the magnet until the beads have formed a tight pellet and the supernatant is clear Without removing the tubes from the magnet carefully transfer the supernatant containing the DNA toa sterile microcentrifuge tube without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet If the supernatant containing the DNA is discolored repeat Steps 6 7 Discard the used ChargeSwitch Magnetic Beads Do not reuse the ChargeSwitch Magnetic Beads For DNA quantitation refer to page 20 Storing DNA Store the purified gDNA at 20 C or use immediately for the desired downstream application Avoid repeated freezing and thawing of gDNA 12 Purification Procedure Automated Protocol Introduction Hardware Requirements This section provides general information for using the ChargeSwitch gDNA Plant Kit catalog no C
23. nd ChargeSwitch Lysis Buffer L18 at 4 C e Store the remaining kit components at room temperature All components are guaranteed stable for 6 months when stored properly The components supplied in the ChargeSwitch gDNA genomic DNA Plant Kit are listed below The amount of reagents provided in the kits are sufficient to perform 96 catalog no CS18000 or 960 catalog no CS18000 10 purifications Note Some reagents in the kit may be provided in excess of the amount needed RNase A 5 mg ml in 10 mM Tris HCl pH 8 5 10 mM EDTA 10 SDS in deionized water ChargeSwitch 10 Detergent D1 ChargeSwitch Lysis Buffer L18 ChargeSwitch Precipitation Buffer N5 ChargeSwitch Wash Buffer W12 ChargeSwitch Elution Buffer E6 10 mM Tris HCl pH 8 5 with 1 mM EDTA Components Amount CS18000 CS18000 10 ChargeSwitch Magnetic Beads 25 mg ml in 4ml 40 ml 10 mM MES pH 5 0 10 mM NaCl 0 1 Tween 20 200 pl 2 ml 10 ml 100 ml 10 ml 100 ml 100 ml 2 x 500 ml 38 5 ml 385 ml 200 ml 2 x 1000 ml 14 5 ml 145 ml Product Qualification Quality Control Each kit is functionally tested to ensure conformance with the most current approved product specifications Current specifications consist of tests for e Bead size charge and binding capacity e Nucleic acid quality and quantity e Buffer turbidity volume and absence of RNases and DNases e Kit packaging and labeling accuracy
24. oll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail E mail tech_support invitrogen com eurotech invitrogen com MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the cont
25. ssays Q33130 Quant iT PicoGreen dsDNA Assay 1 kit 1 ml P7589 E Gel Agarose Gels and DNA Ladders E Gel Agarose Gels are bufferless pre cast agarose gels designed for fast convenient electrophoresis of DNA samples E Gel Agarose Gels are available in different agarose percentages and well formats for your convenience A large variety of DNA ladders is available from Invitrogen for sizing DNA For details on these products visit www invitrogen com or call Technical Support page 23 vi Overview Introduction The ChargeSwitch Technology The ChargeSwitch gDNA Plant Kits allow rapid and efficient purification of genomic DNA gDNA from plant samples including leaves tissues and seeds After preparing the lysates you can purify DNA in less than 15 minutes using the ChargeSwitch Technology without the use of centrifugation or organic solvents For more information about the Charge Switch Technology see below The kits are designed for manual processing of one or multiple samples catalog no CS518000 or for automated processing of multiple samples in 96 well format using liquid handling robots catalog no CS18000 10 The purified DNA is suitable for use in any downstream applications of choice such as PCR restriction enzyme digestion or Southern blotting The ChargeSwitch Technology is a novel magnetic bead based technology that provides a switchable surface which is
26. t for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 2005 2006 Invitrogen Corporation All rights reserved For res
Download Pdf Manuals
Related Search