Invisorb DNA Plant HTS 96 Kit/ C User manual
Contents
1. Please read protocols prior the start of the preparation 50 mg plant material into the QlAwell plate homogenize with Mixer Mill MM 300 Retsch under liquid N add 400 ul Lysis Buffer P Mix inclusive Proteinase K and RNase A to each well of the Qiawell plate Incubate at 65 C for 60 min centrifuge for 10 min at 1 700 x g 4 000 rpm transfer 350 ul of the lysate carefully from the QlAwell plate into the wells of the Prefilter Plate close the Prefilter Plate with a Plate Lid load the whole block Prefilter Plate 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket centrifuge at 1 700 x g 4 000 rpm for 5 min discard the Prefilter Plate add 350 ul Binding Buffer A follow preparing instructions to each well of the 2 ml Collection Plate place the DNA Binding Plate D on top of a new 2 ml Collection Plate Transfer the suspension completely into each well of the DNA Binding Plate D Close the DNA Binding Plate D with the Plate Lid and incubate for 1 min at RT Centrifuge at 1 700 x g 4 000 rpm for 10 min at RT Remove the Plate Lid and discard the filtrate add 500 ul Wash Buffer I to each well of DNA Binding Plate D centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 5 min at RT Remove the Plate Lid and discard the filtrate add 700 ul Wash Buffer Il to each well of DNA Binding Plate D centrifuge at 1 700 x g 4 000 rpm for 5 min at RT disca2. K 2x2ml 4x2ml 24 x2 ml RNase A 2x4ml 4x4ml 24x 4 ml 80 ml 2 x 80 ml 2 x 350 ml Wash Buter final volume 160 ml final volume 2 x 160 ml final volume 700 ml 2x 45 ml 4x45 ml 4x 270 ml Wash Buffer II final volume 2 x 150 ml final volume 4 x 150 ml final volume 900 ml 2 0 ml Collection Plate 4 2x4 12x4 Elution Plate L 2 4 24 Prefilter Plate 2 4 6x4 DNA Binding Plate D 2 4 6x4 Plate Lid 4 8 48 Sealing Foils 2 4 24 Manual 1 1 1 Initial steps Add 56 ml 99 7 Isopropanol Add 56 ml 99 7 Isopropanol Add 630 ml 99 7 to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 75 ml 96 100 ethanol to the bottle Wash Buffer Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix shortly and keep the bottle always firmly closed Add 2 ml ddH gt O to each tube Proteinase K mix thoroughly and store at 20 C to each Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 75 ml 96 100 ethanol to each bottle Wash Buffer Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix shortly and keep the bottle always firmly closed Add 2 ml ddH gt O to each tube Proteinase K mix thoroughly and store at 20 C Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before u
3. C Molecular all rights reserved 1 Invisorb Plant DNA HTS 96 Kit C 0213 Contents Kit content of Invisorb DNA Plant HTS 96 Kit C Symbols Storage Quality control and product warranty Intended use Product use limitation Safety information Product characteristic of the Invisorb DNA Plant HTS 96 Kit C Yield and quality of genomic DNA Sampling and storage of starting material Important points before starting a protocol Preparing reagents and buffers MW 4 NNN OOO A A BW Reagents and equipment to be supplied by user Scheme of Invisorb DNA Plant HTS 96 Kit C Principle and procedure Oo O Protocol DNA extraction from plant material for use on a centrifuge kh Troubleshooting kh A Appendix kh ol Ordering information 2 Invisorb Plant DNA HTS 96 Kit C 0213 Kit contents of Invisorb DNA Plant HTS 96 Kit C Store diluted Proteinase K at 20 C but repeated freezing and thawing will reduce the activity dramatically Dividing the Proteinase K into aliquots and storage at 20 C is recommended Store all other kit components at room temperature RT 2 x 96 DNA extractions 4 x 96 DNA extractions 24 x 96 DNA extractions Catalogue No 7037300200 7037300300 7037300400 Lysis Buffer P 2x 40 ml 4x40 ml 24 x 40 ml 24 mi 2x24 mi 270 mi Binding Buffer A final volume 80 ml final volume 2 x 80 ml final volume 900 ml Elution Buffer 30 ml 60 ml 350 ml Proteinase
4. Check up Wash Buffers for salt precipitates If there are any precipitates solve these precipitates by careful warming 13 Invisorb Plant DNA HTS 96 Kit C 0213 Appendix General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic acid is eluted in will affect it s stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation Avoid vigorous pipeting Pipeting genomic DNA through small tip openings causes shearing or nicki
5. Plate D Elution Plate L in the rotor bucket of the centrifuge Centrifuge for 5 min at 1 700 x g 4 000 rpm Take the DNA Binding Plate D and the Elution Plate L very carefully out of the centrifuge in order to avoid cross contaminations with adherent fluid Discard the DNA Binding Plate D Take the Elution Plate L with the eluted DNA and cover the plate with sealing foil 12 Invisorb Plant DNA HTS 96 Kit C 0213 Troubleshooting Problem probable cause clogged DNA Binding Plate D incomplete lysis of starting material viscous lysates Comments and suggestions increase incubation time with Lysis Buffer P reduce amount of starting material low amounts or none DNA insufficient lysis incomplete elution increase time for lysis reduce amount of starting material or use higher volume of Lysis Buffer P Increase Binding Buffer A to adequate volume Lysis Buffer P Binding Buffer A ratio has to be always 2 1 make sure that the correct amount of ethanol is added to the Wash Buffers make sure that the Buffers are stored correctly Increase incubation time with prewarmed Elution Buffer to 10 min or repeat elution step once again prewarm Elution Buffer up to 80 C problems with downstream applications e g PCR ethanol carryover during elution salt carryover during elution increase centrifugation time for removing of ethanol ensure that Wash Buffer and Wash Buffer Il are at room temperature
6. ail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb DNA Plant HTS 96 Kit C have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of Invisorb DNA Plant HTS 96 Kit C or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany Oo 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 4 Invisorb Plant DNA HTS 96 Kit C 0213 Intended use The Invisorb DNA Plant HTS 96 Kit C is the ideal tools for a rapid and efficient isolation of high quality genomic DNA from up to 50 mg of a wide variety of plant species fresh frozen or dried plant material for instance leaves roots fruits or seeds The protocols for t
7. ase to the environment Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 6 Invisorb Plant DNA HTS 96 Kit C 0213 Product Characteristic of the Invisorb DNA Plant HTS 96 Kit up to 50 mg of a wide up to 6 ug well about 100 min C Azso A 280 variety of plant species inclusive 60 min incubation 1 6 2 0 The Invisorb DNA Plant HTS 96 Kit has been designed for a semi automated preparation of genomic DNA for up to 50 mg of plant sample in 96 well format Due to the high purity the isolated genomic DNA is ready to use for a broad panel of downstream applications PCR Reactions RFLP Analysis Restriction Enzyme Digestion Hybridization O O 0 0 No toxic or hazardous chemicals like chaotropic components are used For the isolation of genomic DNA in high through put format STRATEC Molecular offers the Invisorb DNA Plant HTS 96 Kits for use on different robotic stations For the isolation of DNA from single plant sample STRATEC Molecular offers the Invisorb Spin Plant Mini Kit For further information please contact Phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2907 or your local distributor Yield and quality of genomic DNA The amount of purified DNA in the Invisorb DNA Plant HTS 96 Kit procedure from plant material depends on sample source transport conditions s
8. authorization or implicit licence to practice PCR under any patents held by Hoffmann LaRoche Inc 7 Invisorb Plant DNA HTS 96 Kit C 0213 o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a Suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents This kit should only be used by trained personnel o 9 0 9 0 Preparing reagents and buffers 2 x 96 DNA extractions Add 56 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 75 ml 96 100 ethanol to the bottle Wash Buffer Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix shortly and keep the bottle always firmly closed Add 2 ml ddH gt O to each tube Proteinase K mix thoroughly and store at 20 C 4 x 96 DNA extractions Add 56 ml 99 7 Isopropanol to each Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 75 ml 96 100 ethanol to each bottle Wash Buffer Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix shortl
9. dation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption 5 Invisorb Plant DNA HTS 96 Kit C 0213 Safety Information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bo
10. e bottle of Lysis Buffer P with one tube of Proteinase K and one bottle of RNase A Add 400 ul Lysis Buffer P Mix inclusive Proteinase K and RNase A to each well of the Qiawell plate Incubate at 65 C for 60 min centrifuge for 10 min at 1 700 x g 4 000 rpm 10 Invisorb Plant DNA HTS 96 Kit C 0213 2 Prefiltration Place the Prefilter Plate on top of a 2 ml Collection Plate Transfer 350 ul of the lysate carefully from the QlAwell plate into the wells of the Prefilter Plate Close the Prefilter Plate with a Plate Lid Load the whole block Prefilter Plate 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 700 x g 4 000 rpm at RT Take the Prefilter Plate 2 ml Collection Plate out of the centrifuge Remove the Plate Lid Discard the Prefilter Plate Note You do not need to centrifuge if the lysates have already run through the filter If a Mixer Mill is not available a disruption and homogenization of the plant material must be performed with other methods like with mortar pestle and N or within another kind of mill like Savant with beads or on a Gyrator UniEquip or using a rotor stator homogenizer 3 Binding of the genomic DNA to the DNA Binding Plate D Add 350 ul Binding Buffer A to each well of the 2 ml Collection Plate and mix the solution very well by pipetting several times up and down by using a multichannel pipet Place the DNA Binding Plate D on t
11. g Plate D back to the top of the 2 ml Collection Plate Repeat this washing step 11 Invisorb Plant DNA HTS 96 Kit C 0213 6 Removing of ethanol Take the DNA Binding Plate D 2 ml Collection Plate out of the centrifuge Remove the Plate Lid Place the DNA Binding Plate D onto a clean surface paper towel Empty the 2 ml Collection Plate and dry its upper side with paper Place the DNA Binding Plate D on top of the 2 ml Collection Plate and close it with a Plate Lid Load the whole block DNA Binding Plate D 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket of the centrifuge Centrifuge at maximum speed for at least 15 min at RT to eliminate any traces of ethanol Take the DNA Binding Plate D 2 ml Collection Plate out of the centrifuge discard the Plate Lid and place the plate on a clean paper towel Discard the 2 ml Collection Plate 7 Elution of the genomic DNA Place the DNA Binding Plate D on the top of a Elution Plate L Add 60 ul Elution Buffer directly onto the membrane in each well and incubate for 5 min Close the DNA Binding Plate D with a new Plate Lid and place the whole block DNA Binding Plate D Elution Plate L in the rotor bucket of the centrifuge Centrifuge for 5 min at 1 700 x g 4 000 rpm Remove the Plate Lid Add 60 ul Elution Buffer again directly onto the membrane in each well Close the DNA Binding Plate D with the Plate Lid and place the whole block DNA Binding
12. he isolation and all buffers are optimized for a high yield as well as a high purity All hands on steps are reduced to a minimum For reproducible and high yields appropriate sample storage is essential THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modification of DNA followed by signal detection or amplification Any results generated using the sample preparation procedure in conjunction with any downstream assay should be interpreted with regard to other laboratory findings To minimize irregularities in your results adequate controls for downstream applications should be used Product use limitation The kit is neither suitable for isolation of RNA from plant material as for DNA isolation from cultured or isolated cells tissue samples or blood samples The isolation of simultaneously isolation of bacterial DNA or DNA from fungi and parasites is not validated The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for vali
13. ng One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipeting genomic DNA DNA Yield The amount of purified DNA from the plant material depends on sample source transport conditions storage and age of the sample 14 Invisorb Plant DNA HTS 96 Kit C 0213 Ordering information InviMag Plant DNA Mini Kit KFmL 2437110100 15 purifications InviMag Plant DNA Mini Kit KFmL 2437110200 75 purifications InviMag Plant DNA Mini Kit KF 96 7437300100 1 x 96 preps InviMag Plant DNA Mini Kit KF 96 7437300200 5 x 96 preps Invisorb DNA Plant HTS 96 Kit C 7037300200 2 x 96 preps Invisorb DNA Plant HTS 96 Kit C 7037300300 4 x 96 preps Invisorb DNA Plant HTS 96 Kit C 7037300400 24 x 96 preps Invisorb Spin Plant Mini Kit 1037100200 50 purifications Invisorb Spin Plant Mini Kit 1037100300 250 purifications Possible suppliers for Isopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F 15 Invisorb Plant DNA HTS 96 Kit C 0213 stratecee molecular STRATEC Molecular GmbH Robert R ssle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1C3dC 02 2013
14. op of a new 2 ml Collection Plate Transfer the suspension completely into each well of the DNA Binding Plate D Close the DNA Binding Plate D with the Plate Lid and incubate for 1 min at RT Load the whole block DNA Binding Plate D 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 700 x g 4 000 rpm for 10 min at RT Take the DNA Binding Plate D 2 ml Collection Plate out of the centrifuge Remove the Plate Lid and discard the filtrate Place the DNA Binding Plate D back to the top of the 2 ml Collection Plate 4 First Washing of the DNA Binding Plate D Add 500 ul Wash Buffer I to each well of the DNA Binding Plate D Close the DNA Binding Plate D with the Plate Lid Load the whole block DNA Binding Plate D 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 700 x g 4 000 rpm for 5 min at RT Remove the Plate Lid and discard the filtrate Place the DNA Binding Plate D back to the top of the 2 ml Collection Plate 5 Second washing of the DNA Binding Plate D Add 700 ul Wash Buffer Il to each well of the DNA Binding Plate D Close the DNA Binding Plate D with the Plate Lid Load the whole block DNA Binding Plate D 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 700 x g 4 000 rpm for 5 min at RT Remove the Plate Lid and discard the filtrate Place the DNA Bindin
15. rd filtrate and place the DNA Binding Plate D back on the top of a 2 ml Collection Plate Repeat the wash step remove all waste from the waste tray centrifuge at maximum speed for 15 min to dry the membrane discard 2 ml Collection Plate place the DNA Binding Plate D on the top of a Elution Plate L or of a Microtube Plate add 60 ul Elution Buffer incubate 5 min at RT centrifuge at 1 700 x g 4 000 rpm for 5 min Repeat the elution step Take the DNA Binding Plate D and the Elution Plate L out of the centrifuge Discard the DNA Binding Plate D 9 Invisorb Plant DNA HTS 96 Kit C 0213 Principle and procedure The Invisorb DNA Plant HTS 96 Kit procedure comprises following steps o lysis of sample material o binding the genomic DNA to the membrane o washing and elimination of ethanol o elution of genomic DNA After lysis the DNA binds to the membrane contaminations and enzyme inhibitors are efficiently removed during the following three wash steps and highly purified DNA is eluted in Elution Buffer or water Protocol DNA extraction from plant material for use on a centrifuge Attention The complete disruption and homogenization of the plant samples is absolute essential for isolation of high yield of genomic DNA The disruption procedure the breakage of intercellular matrix and plasma membranes is necessary to release the nucleic acids contained in the plant cell thus inefficient disruption decreases the DNA
16. se mix by inverting several times Add 350 ml 96 100 ethanol to each bottle Wash Buffer I Add 630 ml of 96 100 ethanol to each bottle Wash Buffer Il mix shortly and keep the bottle always firmly closed Add 2 ml ddH gt O to each tube Proteinase K mix thoroughly and store at 20 C Invisorb Plant DNA HTS 96 Kit C 0213 Symbols Manufacturer rc o 4 Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse HKE A Storage All buffers and kit components of the Invisorb DNA Plant HTS 96 Kit C should be stored at room temperature RT and are stable for 12 months under these conditions Room temperature RT is defined as range from 15 30 C Dissolved Proteinase K stored at 20 C is stable for 12 months but repeated freezing and thawing should be avoided Aliquotation and storage at 20 C is recommended Wash Buffer charged with ethanol should be appropriately sealed and stored at room temperature If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb DNA Plant HTS 96 Kit C for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product f
17. stratecee molecular 7 User manual InVisorb DNA Plant HTS 96 Kit C for purification of genomic DNA upto 50 mg of a wide variety of plant species in a 96 well format using acentrifuge 1037300X00 MAN strATEC Molecular GmbH D 13125 Berlin or Instruction for Invisorb DNA Plant HTS 96 Kit for 96 DNA extractions from up to 50 mg of plant material for use on a centrifuge The Invisorb DNA Plant HTS 96 Kit has been designed to purify extremly fast genomic DNA from up to 50 mg plant material using a centrifuge The newly patented technology for isolation of genomic DNA by binding onto the filter surface does not need any hazardous chaotropic buffer components The isolation protocol as well as all buffers is optimized to provide high yield and purity of the extracted genomic DNA The hands on time necessary for the whole procedure is reduced to a minimum Trademarks Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb is a registered trademark of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATE
18. torage and age of the sample Yield and quality of isolated genomic DNA is suitable for any detection system Important indications for the Invisorb DNA Plant HTS 96 Kit The kit can also purify RNA besides DNA For the elimination of RNA if necessary add 20 ul RNase A 10 mg ml to Binding Buffer A follow preparing instructions in Tube A Sampling and Storage of starting material Harvested plant samples can be stored at room temperature for 2 3 hours for short time storage up to one week samples may be stored at 4 C For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing before isolating the DNA should be avoided Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance The PCR method is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the Invisorb DNA Plant HTS 96 Kit cannot be construed as an
19. ttles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the Invisorb DNA Plant HTS 96 Kit procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste has be considered infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb DNA Plant HTS 96 Kit to which they apply are listed below as follows Lysis Buffer P Proteinase K danger danger H319 P305 351 338 H315 319 334 335 P280 305 351 338 310 405 Wash Buffer warning H302 312 332 412 EUH032 P273 H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up P273 Avoid rele
20. y and keep the bottle always firmly closed Add 2 ml ddH gt O to each tube Proteinase K mix thoroughly and store at 20 C 24 x 96 DNA extractions Add 630 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 350 ml 96 100 ethanol to each bottle Wash Buffer I Add 630 ml of 96 100 ethanol to each bottle Wash Buffer Il mix shortly and keep the bottle always firmly closed Add 2 ml ddH gt O to each tube Proteinase K mix thoroughly and store at 20 C Reagents and equipment to be supplied by user o Multichannel pipet with tips Reagents reservoirs for multichannel pipets o Centrifuge for microplates e g Sigma centrifuge 4 15 with plate rotor 2 x 96 or Eppendorf 5804 R 5810 R centrifuge with deepwell plate rotor o Ethanol 96 100 o Mixer Mill 300 and equipment from Qiagen o lsopropanol o The Invisorb DNA Plant HTS 96 Kit C is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for Isopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F 8 Invisorb Plant DNA HTS 96 Kit C 0213 Scheme Invisorb DNA Plant HTS 96 Kit C o o O Oo
21. yield The homogenization means the reduction of the viscosity of the lysate after disruption Contaminating total RNA and other cellular components of high molecular weight are sheared to form a homogenous lysate If the homogenization of the starting material is not done very carefully the yield of genomic DNA purified is reduced significantly It is possible to use a commercially available bead mill or rotor stator homogenizer in combination with or without beads for the disruption and homogenization of the starting material Alternatively the starting material can reduced to a fine powder in liquid nitrogen using a mortar and pestle Rotor stator homogenizers and bead mills simultaneously disrupt and homogenize the plant material whereas plant tissue are only disrupted using a mortar and pestle and a separate homogenization step should be performed Note Elution with prewarmed Elution Buffer up to 80 C will also increase the final yield 1 Disruption Homogenization and Lysis of the plant material in a Mixer Mill Note Its recommended to use a mixer mill for the simultaneous homogenization of 96 plant samples The Mixer Mill from Retsch works in a 96 well format but only in combination with a special adapter from Qiagen Therefore the use of QlAwell plates not provided is necessary Transfer up to 50 mg plant material into the QlAwell plate not provided and homogenize with Mixer Mill MM 300 Retsch under liquid No Mix on
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