illustra triplePrep Kit ブックレット
Contents
1. Table 3 Sample Type Rat Liver HeLa Cells Sample input amount 1 20 mg 0 3 5 million Genomic DNA yield 1 3 ug per mg 4 12 ug per million tissue cells Total RNA yield 3 7 ug per mg 10 15 ug per million tissue cells Protein yield 80 160 ug per mg 100 200 ug per tissue million cells Genomic DNA purity sel A gt co gol Genomic DNA size o kb Total RNA purity 5 9 A o A so Total RNA quality RIN 8 0 10 Total RNA auality 1 5 2 5 285 185 Time preparation 45 60 minutes excluding lysis Note The mini column with orange o ring has a binding capacity of 20 ug for genomic DNA About 15 ug of DNA can be eluted on 1st elution if 20 ug or more DNA is present in the crude lysate DNA yield will not be increased when more than 20 ug of DNA is processed The mini column with pink o ring has the binding capacity of 60 ug of total RNA About 45 ug of total RNA can be eluted on 1st elution if 60 ug or more total RNA is present in the flowthrough RNA yield will 17 not be increased when more than 60 ug of RNA is processed Loading the system with sample containing more nucleic acids than the columns can bind will not increase yield and sometimes may even lower the quality and purity of purified nucleic acids For example around 15 ug of genomic DNA and 45 ug of total RNA usually can be isolated from 10 mg of rat liver which contains 20 25 ug of genomic DNA and 50 70 ug of total RNA However DNA2. e Mechanical agitation device 33 A common laboratory scale mechanical method for cell disruption that uses glass or ceramic beads and a high level of agitation It is normally used for samples are tough to disrupt yeast spores and micro algae It has moderate efficiency The major drawbacks for a mechanical agitation device are it has large variability in product yield and purity and occasional problems with foaming and sample heating At the lowest levels of the technology the beads are added to the cell suspension in a tube and the sample is mixed on a common laboratory vortex mixer This process works for easily disrupted cells is inexpensive and multiple samples can be conveniently processed The more sophisticated level bead based methods use a closed container holding the sample and the beads with an electric motor to provide vigorous agitation When larger samples are processed some form of cooling is provided typically liquid CO as the sample heats Significantly due to the extreme agitation Another configuration uses an inert rapidly rotating rotor in a small container containing the cells and beads e Mortar and pestle style grinders Grinding with mortar and pestle is the most commonly used technique for disruption of animal tissue The technique tends to be a gentle cell disruption method significantly gentler than e g sonication However this technique is laborious slow and usually inefficient It gener
3. Table 2 Step 1 Sample homogenization and lysis 2 DNA binding 3 DNA wash 1 4 DNA wash 2 5 DNA elution 6 RNA binding 7 DNase treatment optional Comments Tissue and cells are lysed in presence of Lysis buffer type 15 with 2 Mercaptoethanol Chaotropic salt in Lysis buffer type 15 promotes the binding of DNA to the silica membrane Lysis buffer type 15 removes contaminants from membrane bound DNA The ethanolic Wash buffer type 6 removes residual salts and dries the silica membrane at the same time DNA is eluted in a low ionic strength buffer Acetone promotes the binding of total RNA to the silica membrane in the presence of chaotropic salt in Lysis buffer type 15 On column DNase digestion removes residual genomic DNA bound to the silica membrane 14 Component Lysis buffer type 15 add 196 2 Mercaptoethanol illustra mini columns DNA orange o ring Lysis buffer type 15 Wash buffer type 6 Elution buffer type 5 Acetone user supplied illustra mini columns RNA pink o ring DNase I and DNase reaction buffer type 1 Step Comments Component 8 RNA wash The ethanolic Wash Wash buffer type 6 buffer type 6 removes residual salts and dries the silica membrane at the same time 9 RNA elution RNA is eluted in RNase Elution buffer type 9 free water 10 Protein Acid and salt based Protein precipitation precipitation Protein precipitation buffer type 1 buffer
4. d Proceed immediately to Step 6 to isolate RNA Total RNA isolation 6 RNA binding a Place a new mini column pink o ring in q new Collection tube b Add 350 ul of Absolute Acetone to the flowthrough from Step 2d Mix well by pipetting up and down several times Transfer the entire mixture to the mini column c Spin for 1 minute at 11 000 x g pd min 11 000 xg x d Save the flowthrough for protein isolation atroom temperature Refer to Step 10 for further processing e Transfer the column to a new Collection tube provided Proceed to Step 7 or Step 8 as required 7 DNase treatment optional Kit s mini column with orange o ring has the binding capacity of 20 ug DNA Crude lysate containing more than 20 ug DNA for example when 10 mg rat liver or 3 million HeLa Cells are processed may require use of DNase to remove residual genomic DNA in the isolated RNA l a In a RNase free microcentrifuge tube add MU 10 ul of reconstituted DNase I 66 20 ul of lii DNase reaction buffer type 1 and 70 ul RNase free water Mix by flicking the tube Do not vortex b Apply 100 ul of diluted DNase directly on the L center of membrane of the column Incubate for 10 minutes at room temperature Proceed to Step 8 8 RNA wash 500 ul Wash buff a Add 500 ul of Wash buffer type 6 m to the l column b Spin for 1 minute at 11 000 x g Discard the Collection tube a
5. and RNA yield will not be doubled if 20 mg of rat liver is processed due to the capacity of DNA and RNA column being reached Excess DNA that does not bind to the 1st column will flow through into the RNA binding and elution steps If user has large amounts of tissue or cells it is highly recommended to split the samples and process as several samples for better quality and yield of nucleic acids As protein is precipitated out without binding to any columns yield of protein is relatively linear to sample input amount i e If 100 ug of protein can be isolated from 1 mg of certain type of tissue then 2000 ug of protein is expected from 20 mg of this type of tissue 18 4 4 Typical output Table 4 Typical yields and purities of genomic DNA total RNA and total Protein from different tissue and cells Cell HeLa NIH 3T3 CHO K1 HEK 293 Cell input 1 1 1 1 amount million DNA Vield ug 8 5 9 5 4 4 91 Purity 19 1 9 19 19 A280 RNA Yield ug 13 7 7 9 6 6 10 1 Purity Aygo 21 21 2 0 20 A280 Protein Yield ug 139 127 77 139 Tissue Liver Brain Heart Kidney Lung Spleen Tissue input 10 10 10 10 10 10 amount mg Disruption Easy Easy Hard Med Med Med difficulty DNA Yield ug 14 6 6 8 9 8 17 0 15 5 18 8 Purity Azgo 1 9 18 19 19 18 18 A280 RNA Yield ug 44 5 5 2 9 AA 8 8 18 9 Purity Azgo 21 2 0 19 19 2 0 24 A280 Need DNase Yes No No Yes Yes Yes Protein Yield pg 1460 746 923 897 592 510 Yield and purities of
6. 12 months D f Add 350 ul of Lysis buffer type 15 containing 2 Mercaptoethanol g Pipet up and down to resuspend the cell pellet h Homogenize by passing lysate at least 5 times through a DNase amp RNase free 20 gauge needle fitted with 1 ml sterile syringe f Proceed immediately to Step 2 or store at 80 C for up to 6 months Homogenized sample in Lysis buffer type 15 containing 2 Mercaptoethanol may be stored at 80 C for up to 6 months E Cells grown in monolayer in a 6 well plate can be lysed directly Remove media and guickly wash cells with 500 ul cold PBS Remove PBS and add 350 ul of Lysis buffer type 15 28 containing 2 Mercaptoethanol per well of the 6 well plate Incubate for 1 minute at room temperature Pipet up and down several times to resuspend and homogenize using a needle amp syringe as above Genomic DNA isolation 2 DNA binding a Place a new mini column orange o ring into a 2 ml Collection tube provided b Transfer the homogenized lysate from Step 1 350 ul to the column c Spin for 1 minute at 11 000 x g 1min 11 000 xg d Save the flovvthrough for RNA and Protein lt isolation at room temperature Refer to Step 6 or Step 10 for further processing e Transfer the column to a new 2 ml Collection tube provided Proceed to Step 3 Ra Should the column clog repeat Step 2c 1 2 times If it still clogs initial sample amount should be reduced WN U
7. 4 C on arrival to extend its shelf life Once reconstituted in RNase free water aliquot and store DNase at 20 C Avoid vigorous mixing of the enzyme All other kit components should be stored at room temperature 20 25 C Storage at lower temperatures may cause precipitation of salts 2 3 Expiry For expiry date please refer to outer packaging label DNase reconstituted in RNase free water is stable for 6 months when stored at 20 C 3 Components 3 1 Kit contents Identification Pack Size Cat No Lysis buffer type 15 blue colored cap Wash buffer type 6 yellow colored cap Elution buffer type 5 silver colored cap Elution buffer type 9 pink colored cap Protein precipitation buffer type 1 green colored cap DNase I black colored cap 10 purifications 50 purifications sample pack 28 9425 44 75 ml 75 ml Remove Remove aliquot and add aliquot and add 2 Mercaptoethanol 2 Mercaptoethanol to aliquot before use use 12 ml 12 ml Add 48 ml Ethanol Add 48 ml Ethanol before 1st time before 1st time to aliguot before use use 12 ml 12 ml 7 5 mi 7 5 ml 2x 17 5 ml 2x 17 5 ml 1 viql 1 vial DNase reaction buffer type 1 1 4 ml 1 4 ml red colored cap 4 illustra mini columns DNA 10 50 _ orange o ring ch illustra mini columns 10 50 RNA pink o ring Collection tubes 1x50 4x50 Refer to the Certificate of Analysis for a complete list of kit component
8. Glassware should be oven baked for at least 2 hours at 250 C before use Wipe down laboratory surfaces with an alcohol wipe To preserve stability always keep the isolated total RNA on ice or frozen at 80 C for long term storage Purified RNA concentration should be determined by UV spectrophotometer and through comparison with a known standard by denatured agarose gel electrophoresis The reliable range of UV spectrophotometric data should be determined for individual spectrophotometers Generally for spectrophotometers with a 1 cm path length A s readings should lie between 0 1 and 1 0 and appropriate dilutions 4 to 40 ng ul should be analyzed For NanoVue spectrophotometers absorbance readings between 1 and 10 provide more reliable results The UV spectrophotometric ratios A A provide information regarding the purity of RNA A purity ratio of 1 9 to 2 1 indicates that the RNA is pure for all standard molecular biology applications If the ratio is lower than 1 9 the purified RNA might contain some protein impurities 1 OD unit A is equivalent to approximately 40 ug ml RNA Yield Az x 40 ug ml x 0 1 ml the total ug of purified RNA in the sample 39 6 5 Protein handling and quantification Protein isolated by triplePrep Kit can be directly applied to 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE study 2 D DIGE is a method that labels protein samples prior to 2 D electrophoresis enabli
9. PAGE or Western Blot by mixing with 1 volume of 2096 SDS Add appropriate sample loading buffer i e 1 volume of 2x Laemmli buffer and incubating at 70 C for 10 minutes before gel loading 40 6 6 DNA contamination removal in eluted RNA The on column DNase treatment procedure provided in protocol Step 7 should be followed if DNA contamination in RNA is expected The supplement protocol below is for DNA contamination removal in RNA after column purification a In a RNase free microcentrifuge tube add 100 ul of RNA contaminated with DNA 10 ul of DNase reaction buffer type 1 and 5 ul of reconstituted DNase I 88 Mix by flicking the tube Do not vortex Incubate for 20 minutes at room temperature b Add 230 ul of Lysis buffer type 15 B and 350 ul of Absolute Acetone Mix well by pipeting up and down several times Transfer the entire mixture to a new mini column c Spin for 1 minute at 11 000 x g Transfer the column to a new Collection tube d Add 500 ul of Wash buffer type 6 mm to the column Spin for 1 minute at 11 000 x g Discard the Collection tube and its contents WN Please check that no residual flowthrough remains in the column outlet If any remains empty the Collection tube put the column back for re centrifuge e Transfer the column to a fresh RNase free 1 5 ml microcentrifuge tube Add 100 ul Elution buffer type 9 m to the center of the column 50 ul or less of Elution buffer type 9 can be used for achievi
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11. genomic DNA total RNA and total Protein may vary depending on nature and condition of input sample and user 19 Fig 3 Genomic DNA from 1 million HeLa cells was isolated with triplePrep kit and evaluated using 0 896 agarose gel electrophoresis 2 ul of genomic DNA 100 ul elution was loaded in each well ell Lamda DNA Ladder 23 kb Fig 4 Total RNA from 10 mg rat liver was isolated with triplePrep kit and evaluated using the Agilent 2100 bioanalyzer The triplePrep kit produces high quality RNA the 28 s 18 s 2 2 RNA Integrity Number RIN 9 8 J hi Lie 25 200 500 1000 2000 4000 nt 20 Fig 5 Total protein from 10 mg rat liver was isolated with triplePrep kit and evaluated using a Western Blot B Actin and b 2 D DIGE Western blot Rat liver B Actin 42 kDa b Overlaid image yellow of triplePrep green and reference gel red As seen more spots are detected by triplePrep method green spots 21 5 Protocol Use of icons The key below describes the purpose of the icons used throughout the protocol booklet PN This icon is used to highlight particularly critical steps within the protocol that must be adhered to If this advice is not followed it will have a detrimental impact on results Eg This icon is used to highlight technical tips that will enhance the description of the step These tips may indicate areas of flexibility in the protocol or give a recomm
12. type 1 11 Protein wash Water solubilizes and Water user supplied removes acid and salt from protein pellet 12 Protein re Urea and detergent in 2 D DIGE buffer user suspension 2 D DIGE buffer ensure supplied complete protein pellet solubilization 1st time users of triplePrep should follow the detailed protocol in section 5 The guick reference protocol is for experienced users only It is highly recommended that when processing a type of tissue or cell for the first time users should start with 5 mg tissue or 1 million cells Alter the input sample amount depending on initial results As shown in Fig 2 sample is lysed in Lysis buffer type 15 which contains large amounts of chaotropic salts This lysis buffer immediately inactivates DNase RNases and proteases which are present in virtually all biological materials and creates appropriate binding conditions that favor adsorption of DNA to the silica membrane The DNA RNA and protein especially RNA within tissue and cells are vulnerable to attack by both endogenous enzymes and those in the general laboratory environment 15 Use fresh material whenever possible Where this is not possible it is important that samples are flash frozen in liguid nitrogen immediately and stored at 80 C or stored in a stabilizing agent Samples should always be stored at 80 C Never allow tissues to thaw before addition of Lysis buffer type 15 and disrupt samples in liquid nitrogen i
13. 15 p For each sample being processed aliquot 350 ul of Lysis buffer type 15 to a fresh DNase amp RNase free tube and add 3 5 ul of 2 Mercaptoethanol Prepare a fresh aliquot based on total number of samples being processed each time the triplePrep kit is used 2 Mercaptoethanol is not stable in Lysis buffer type 15 Do NOT add 2 Mercaptoethanol directly to the kit bottle of Lysis buffer type 15 Lysis buffer type 15 without 2 Mercaptoethanol is required during the protocol Wash buffer type 6 tw Add 48 ml Absolute Ethanol to Wash Buffer type 6 before use Mix by inversion and indicate on the label by ticking the box that this step has been completed Store upright and at room temperature 20 25 C DNase Add 540 ul of RNase free water to the DNase vial and incubate for 1 minute at room temperature Aliquot and store at 20 C Reconstituted DNase is stable for 6 months when stored at 20 C Avoid vigorous mixing of the enzyme Do NOT vortex Invert vial to mix 23 Protein pellet resuspension buffer user supplied The 2 D DIGE buffer is a suitable buffer to resuspend protein pellets obtained with the illustra triplePrep kit Preparation instruction is shown in Table 5 Table 5 2 D DIGE buffer preparation 100 ml Scale Final concentration Distilled water 40 ml Urea 42 09 7M Thiourea 15 2g 2M Tris HCI 0 36 g 30 mM CHAPS 3 3 Cholamidopropyl 409 496 dimethylammoniol 1 propanesulfonate Mix wel
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15. DIGE buffer are supplied by GE Healthcare see Section 5 1 page 23 for preparation details Protein resuspended in 2 D DIGE buffer can be used for SDS PAGE or Western Blot by mixing with 1 volume of 20 SDS Sodium dodecyl sulfate Add appropriate sample loading buffer i e 1 volume of 2x Laemmli buffer and incubate at 70 C for 10 minutes before gel loading Optional Phosphate buffered saline PBS if processing fresh cultured cell lines Optional Liquid nitrogen Optional Protein concentration determination reagents e g 2 D Quant Kit supplied by GE Healthcare Disposables e 1 5 ml DNase 6 RNase free microcentrifuge tubes e DNase amp RNase free pipet tips 3 3 Equipment needed e Microcentrifuge that accommodates 1 5 ml microcentrifuge tubes and can spin at 16 000 x g or higher e Vortex mixer e Rotor stator homogenizer or mortar and pestle e Optional Nuclease free 20 gauge needle and syringe 1 ml if using mortar amp pestle or for cell lysate homogenization 4 Description 4 1 Introduction The illustra triplePrep Kit is designed for the rapid extraction of genomic DNA total RNA and total denatured protein from a single undivided sample starting from as little as 1 mg of animal tissue or 0 3 million cultured mammalian cells The protocols for extraction from both tissues and cell lines utilize the same buffers Although the protocols are rapid they have been designed to minimize shearing of genomic DNA mai
16. GE Healthcare See back cover for guick reference protocol card illustra triplePrep Kit For the isolation of genomic DNA total RNA and total denatured protein from a single undivided sample of tissue or cultured cells Product booklet Code 28 9425 44 Page finder Lar Legal Handling 2 1 Safety warnings and precautions 2 2 Storage 2 3 Expiry Components 3 1 Kit contents 3 2 Materials to be supplied by user 3 3 Equipment needed Description 4 1 Introduction 4 2 The basic principle 4 3 Product specifications 4 4 Typical output Protocol 5 1 Preparation of working solutions 5 2 Protocol for isolation of DNA RNA and Protein from tissue or cells Appendices 6 1 Tissue homogenization considerations 6 2 Estimation of cell density 6 3 DNA handling and quantification 6 4 RNA handling and quantification 6 5 Protein handling and quantification 6 6 DNA contamination removal in eluted RNA 6 7 RPM to RCF calculation 6 8 Troubleshooting guide 6 9 Related products O O NN Ot On coc A PrP PrP PR ON o N N WU N 25 35 35 37 38 39 40 41 42 43 51 7 References 55 Ouick Reference Protocol Card Back Cover Tear off sheet containing protocols for the experienced user isolating DNA RNA and protein from tissue or cultured cells 1 Legal Product use restriction The components of the illustra triplePrep Kit have been designed
17. Suboptimal performance of RNA in downstream experiments Possible cause Suggestions Carry over of Ethanol Do not let the flowthrough touch the or salt column outlet after the wash with Wash buffer type 6 Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic buffer Wash buffer type 6 completely e Check if Wash buffer type 6 has been equilibrated to room temperature before use Washing at lower temperatures lovvers efficiency of salt removal by Wash buffer type 6 Store isolated RNA e Eluted RNA should always be kept on properly ice for optimal stability since trace contaminations of omnipresent RNases will degrade the isolated RNA For storage freeze at 80 C Problem Protein pellet can not be resuspended Possible cause Suggestions Wrong protein 2 D DIGE buffer is the best tested buffer resuspension buffer to solubilize precipitated protein used Incomplete salt e Follow the protocol break the protein removal during pellet pellet with pipet tip during water wash wash step for effective removal of salts 50 6 9 Related products A full range of Molecular Biology reagents can be found in the GE Healthcare web site and in the catalogue Application Genomic DNA purification Product illustra tissue and cells genomicPrep Mini Spin Kit illustra tissue and cells genomicPrep Mini Spin Kit illustra tissue and cells genomic Midi Flow Kit illustra bacteri
18. a genomicPrep Mini Spin Kit illustra bacteria genomicPrep Mini Spin Kit illustra blood genomicPrep Midi Flow Kit illustra blood genomicPrep Mini Spin Kit illustra blood genomicPrep Mini Spin Kit 51 Product Code 29 9042 75 29 9042 76 28 9042 75 28 9042 58 28 9042 59 28 9042 61 28 9042 64 28 9042 65 Pack Size 50 preps 250 preps 25 preps 50 preps 250 preps 25 preps 50 preps 250 preps Application RNA purification For PCR or RT PCR Product illustra RNAspin Mini RNA Isolation Kit illustra RNAspin Mini RNA Isolation Kit illustra RNAspin Mini RNA Isolation Kit illustra RNAspin Midi RNA Isolation Kit illustra RNAspin 96 RNA Isolation Kit illustra RNAspin 96 RNA Isolation Kit Hot Start Master Mix lllustra Hot Start Mix RTG lllustra Hot Start Mix RTG illustra PuReTagTM Ready To Go PCR Beads illustra PuReTaq Ready To Go PCR Beads 52 Product Code 25 0500 70 25 0500 71 25 0500 72 25 0500 73 25 0500 74 25 0500 75 25 1500 01 29 9006 46 29 9006 53 27 9557 01 27 9557 02 Pack Size 20 preps 50 preps 250 preps 20 preps 2 x 96 preps 4 x 96 preps 100 reactions 0 5 ml 100 rxns 0 2 ml 96 rxns 96 reactions in 0 2 ml tubes plate 5x96 reactions in 0 2 ml tubes plate Application Product For PCR or FideliTagTM PCR RT PCR Master Mix Plus 2x1 FideliToqTH PCR Master Mix P
19. able for at least 30 minutes at room temperature Experienced users may also simultaneously purify DNA and RNA first to save time and ensure RNA guality when DNase treatment is not needed The illustra triplePrep Kit conveniently provides a flexible lysis buffer for the long term storage of samples avoiding the need for costly stabilization reagents Following sample disruption samples can be stored in Lysis buffer type 15 at 80 C for 6 months or up to 4 hours at room temperature 1 Sample Homogenization Fig 1 amp Lysis 2 DNA binding L 3 DNA wash 1 6 RNA binding 4 DNA wash 2 5 DNA elution 7 DNase treatment 10 Protein precipitation optional 8 RNA vvash DNA Purification R 12 Protein resuspension 9 RNA elution Protein Isolation RNA Purification The kit contains sufficient reagents and columns for 50 28 9425 44 purifications of DNA RNA and protein from 5 mg animal tissue or 1 million cultured cells 11 4 2 The basic principle Use of the illustra triplePrep Kit involves the following steps Fig 2 1 Sample S lysis v DNA binding v DNA wash 1 v DNA wash 2 RNA and Protein in flowthrough wv DNA elution v RNA binding 12 RNA binding continued v DNase treatment optional v RNA wash Protein in flowthrough v RNA elution v Protein precipitation v Protein wash v Protein resuspension 13
20. ammalian cells grown inside a 25 cm flask to confluence yield approximately 2 5 x 106 cells per flask Visit Corning web site at http catalog2 corning com Lifesciences If a 25 cm flask was used add 2 5ml of PBS Make sure cells are completely resuspended without any visible clumps 4 Add 10 ul of resuspended cells to two chambers of hemocytometer separately under a small coverslip making sure the solution spreads completely under the coverslip by capillary action Place the hemocytometer under a light microscope focus the cells using lowest magnification and begin counting cells only at the four corner squares and the middle square in both chambers of the hemocytometer grid 3 Count all cells except those touching the middle lines at the bottom and right Aim to have 50 100 cells per square grid If cell count is gt 150 grid it is advisable to dilute the cells clean the hemocytometer and re count cells 37 Add the number of cells in a total of ten chambers five from each side and multiply by 1000 to give the number of cells ml of PBS 6 3 DNA handling and quantification DNA should be treated with care Elution buffer type 5 contains EDTA to inhibit the activity of any trace amount of DNase DNA can be kept at room temperature for several hours or 4 C for several days To preserve stability Keep the isolated DNA frozen at 20 C or 80 C for long term storage Purified DNA concentration should be determined b
21. ates heat so cooling is necessary The grinders consist of a grinding container mortar and a pestle The cells are sheared between the wall of the container and the edge of the pestle This method requires careful attention to conditions such as force and cooling Tissue shall be snap frozen in liquid nitrogen and disrupted without thawing Extra steps of homogenization are required after disruption The illustra triplePrep Kit recommends use of a needle and syringe for homogenization after grinding 36 6 2 Estimation of cell density It is recommended not to exceed 5 x 106 cells when purifying genomic DNA from mammalian cultured cells DNA yields drop when silica columns begin to experience clogging seen at 1 x 107 cells Cell density should be estimated using an automated cell counter e g Coulter or individual cells should be counted under a microscope using a standard hemocytometer Hausser Scientific VWR 1483 If cell count exceeds 5 x 10 cells ml cells should be split into two DNA preparations Below are guidelines for measuring cell density using a hemocytometer 1 Clean a hemocytometer and the short coverslip thoroughly and wipe clean with ethanol If working with adherent cells trypsinize the cells and wash once with PBS Otherwise pellet the cells 5000 rom for 1 minute such as those grown in suspension Resuspend cells in appropriate volume of PBS to yield roughly 1 x 106 cell ml For example m
22. ded in solution and free of any visible clumps It is highly recommended that tissue is disrupted and homogenized as guickly as possible while minimizing any temperature increase If using any instrument for tissue disruption and homogenization ensure that you are familiar with the operating procedure by referring to its user manual and handbook Some instruments may require use of greater than 350 ul Lysis buffer type 15 Please use the volume recommended by your instruments Load only 350 ul of crude lysate to genomic DNA mini column e Rotor stator homogenizer A rotor stator homogenizer can simultaneously disrupt and homogenize most animal tissues and cells in less than 60 seconds It has moderate efficiency It generates negligible heat during operation The major drawback for a rotor stator homogenizer is it does not work well with difficult to lyse samples The cell tissue suspension is drawn into the apparatus by a rotor sited within a stator hollow tube The suspension is then centrifugally thrown outward to exit through slots or holes on the tip of the stator When the rotor spins it sucks the sample up and cuts it while it is rotating When the sample is pushed out of the slots it is cut further Outside of the slots the sample meets a pressure differential which shears the cells even further Most laboratory rotor stator homogenizers are top driven at a speed of 8000 60 000 rpm The cell Suspension is recycled several times
23. developed and sold for research purposes only They are suitable for in vitro use only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is the responsibility of the user to verify the use of illustra triplePrep Kit for a specific application as the performance characteristic of this kit has not been verified to any specific species GE imagination at work and GE monogram are trademarks of General Electric Company illustra PuReTaq Ready To Go HyBond Deep Purple Rainbow and NanoVue are trademarks of GE Healthcare companies All third party trademarks are the property of their respective owners 2008 General Electric Company All rights reserved First published Oct 2008 All goods and services are sold subject to the terms and conditions of sales of the company within GE Healthcare which supplies them A copy of the terms and conditions is available upon reguest Contact your GE Representative for the most current information http www gelifesciences com GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 2 Handling 2 1 Safety warnings and precautions Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals All chemicals should be considered as potentiall
24. endation to obtain optimum performance of the kit This icon is used to highlight convenient points that the protocol can be stopped Important notes e External factors sample nature storage condition etc may affect the auality and yield of isolated genomic DNA total RNA and total protein e 1st time users of triplePrep should follow the detailed protocol in Section 5 The quick reference protocol is for experienced users only e Do NOT overload the columns with too much starting material or too much nucleic acid rich lysate It is highly recommended that when processing a type of tissue or cell for the first time users should start with small amount of sample input 5 mg tissue or 1 million cells Alter the input sample amount depending on initial results e t is highly recommended that users determine the amount of starting material especially during the first time processing certain types of sample with triplePrep kit 22 e t is important to load only 350 ul of initial homogenized sample in Lysis buffer type 15 onto the genomic DNA mini column The RNA binding and protein precipitation have been optimized based on 350 ul of lysate being used e Perform all steps of the procedure including incubation and flowthrough storage at room temperature unless instructed otherwise 5 1 Preparation of working solutions See Sections 3 2 amp 3 3 for Materials amp Eguipment to be supplied by user Lysis buffer type
25. ential for efficient nucleic acid preparation that all the nucleic acid contained in the sample is released from the cells by disruption and that the viscosity of the sample is reduced by homogenization Following homogenization the tissue should be uniformly suspended in solution and free of any visible clumps Minimize any temperature increases at this stage A rotor stator homogenizer is recommended due to its fast tissue disruption speed high recovery and consistency See section 6 1 for further information on tissue homogenization Fresh animal tissue should be either processed immediately or Snap frozen in liquid nitrogen The triplePrep Kit is compatible with available stabilization reagents but RNA yield may be impacted The illustra triplePrep Kit conveniently provides a flexible solution to the long term storage of samples avoiding the need for costly Stabilization agents Following sample disruption samples can be stored in Lysis buffer type 15 at 80 C for 6 months or up to 4 hours at room temperature Frozen tissue should not be thawed but processed immediately in Lysis buffer type 15 containing 2 Mercaptoethanol during simultaneous mechanical disruption e g with a rotor stator homogenizer This ensures that the RNA is not degraded by RNase before the preparation has started 25 Fresh cultured cells can be lysed after pelleting or lysed directly on the culture plate for detailed protocol see note in Step 1 iii Fro
26. f possible DNA is bound to the 1st mini column orange o ring in the presence of a chaotropic solution Contaminants are removed from DNA with Lysis buffer type 15 and Wash buffer type 6 DNA is eluted with Elution buffer type 5 Total RNA and protein do not bind to the 1st mini column and are collected in the flowthrough Acetone is added to the flowthrough and total RNA is bound to the 2nd mini column pink o ring An optional on column DNase digestion step may be undertaken at this point see Step 7 Page 31 for details of when to perform a DNase digestion Contaminants are removed from the total RNA with Wash buffer type 6 and total RNA is eluted with Elution buffer type 9 Purified RNA should be treated with care because RNA is very sensitive to trace contaminations of RNase often found on general labware fingers and dust Wear gloves at all times during the preparation Change gloves frequently Wipe down all laboratory surfaces with alcohol wipes regularly To preserve stability keep the isolated total RNA frozen at 80 C for long term storage Protein does not bind to the RNA mini column and is collected in the flowthrough A rapid protein precipitation step is undertaken and the protein sample can then be washed and resuspended in 2 D DIGE buffer for a 2 D DIGE study or SDS PAGE and Western Blotting 16 4 3 Product specifications Figures shown are expected performance of illustra triplePrep Kit shown in Table 3
27. ial for each sample If necessary split the tissue between two preparations Incomplete e Make sure cells are completely resuspension of resuspended by pipetting up and down cell pellet in Lysis and vortexing Use needle and syringe buffer type 15 for lysate homogenization Check for absence of cell pellet 43 Problem Low genomic DNA yield continued Possible cause Suggestions Incorrect cell numbers Use 1 2 million cells for optimal recovery used as starting e Do not exceed 5 million cells as yields material may drop See section 6 2 for Estimation of Cell Density Problem Poor guality of isolated genomic DNA Possible cause Suggestions Too much tissue or too Ensure correct amounts used many cells used per sample Failed to perform first e Repeat isolation taking care to complete wash with Lysis buffer all steps type 15 Problem Restriction enzymes fail to cut isolated genomic DNA Possible cause Suggestions Failed to perform all e Repeat isolation taking care to complete wash steps all steps Remove any residual Ethanol after column wash before elution Sub optimal digestion Use 50 100 U of high concentration conditions used restriction enzyme per ug of genomic DNA in the digest Set up reaction in 50 100 ul volumne and incubate the digest overnight 16 hours AA Problem RNA is degraded no RNA obtained Possible cause RNase contamination Suggestions e Create an RNase free working env
28. ironment Wear gloves during all steps of the procedure and change gloves freguently Use of RNase free disposable polypropylene tubes is imperative Keep tubes closed whenever possible during the preparation Glassware should be ovenbaked for at least 2 hours at 250 C before use Problem Poor RNA guality or yield Possible cause Reagents not prepared stored or applied properly Suggestions e Reagents not properly stored e Add the indicated volume of nuclease free water to DNase I vial and mix Add the indicated volume of Absolute Ethanol to Wash buffer type 6 and mix e Reconstitute and store lyophilized DNase according to instructions given in section 5 1 e Sample and reagents have not been mixed completely Vortex vigorously after each reagent has been added except for DNase e Absolute Acetone was not added after lysis Binding of RNA to the silica membrane is only effective in the presence of Absolute Acetone 45 Problem Poor RNA guality or yield continued Possible cause Suboptimal elution Sample material Suggestions e Reconstitute and store lyophilized DNase according to instructions given in section 5 1 e Store other kit components at room temperature Storage at low temperatures may cause salt precipitation e Keep bottles tightly closed in order to prevent evaporation or contamination e Be sure that all of the water Elution buffer type 9 gets into contact with the silica
29. l do not heat over 37 C Adjust final pH to 8 5 8 0 9 0 and adjust to final volume with distilled water Dispense into 5 ml aliquots and store at 20 C with a 3 month expiry date if using for 2 D DIGE study Protein resuspended in 2 D DIGE buffer can be used for 2 D DIGE directly Protein resuspended in 2 D DIGE buffer can be used for SDS PAGE Western Blotting by mixing with 1 volume of 2096 SDS Add appropriate sample loading buffer i e 1 volume of 2X Laemmli buffer and incubating at 70 C for 10 minutes before gel loading 596 SDS or Laemmli buffer alone are not recommended for protein resuspension due to the significant reduction in protein solubilization efficiency The 2 D Ouant Kit supplied by GE Healthcare is recommended for protein concentration quantification Please note 2 D DIGE buffer is not compatible with Bicinchoninic Acid BCA or Bradford protein quantification assays Alternatively 7 M Urea can be used for protein resuspension and further diluted to less than 3 M to be compatible with BCA or Bradford assays 24 5 2 Protocol for isolation of DNA RNA and Protein from tissue or cells Sample homogenization 8 lysis The complete disruption and homogenization of animal tissue is critical Animal tissues are often solid and must therefore be broken up mechanically as well as lysed Depending on the disruption method the viscosity of the lysed sample has to be reduced further for optimal results It is ess
30. lthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shiniuku ku Tokyo 169 0073 Japan For contact information for your local office please visit www gelifesciences com contact GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK http www gehealthcare com lifesciences imagination at work 28940931 PL Rev AB 12 2008 v as ads p o umop pup dn jadig P x 000 TI UIW T IM ulu s 2 6 d42 4ojjng uonni3 IA oor mm Ja4nq 3910 0 2 IN 00s iri os db uonn YNY 6 uoisuodsnsou UID OJJ 21 6 x 000 Ilullu I ogn3 o6nyji3u92042iu S adX1 s yinq uonnjg ir oor lq ssod sp Jupypusadns JW ST 99JJ OSDNNU O UWNJOJ uonnje YNG S Uonu sp anoway 6 x 000 91 UIT UBNOJU1MOJJ ayy papas p 5 x 000 TT Bunadid q uie3o4d 9 adX1 s unq usoM iri oos gn qa aqn BnJH1u 30O43ltu osiedsip Je1pM paj nsip uu T GE USDM YNY 8 JW ST 99JJ OSDNO 01 UWNJOJ JAJSUDJJ USDM TT U NOJU1MOJJ y pJDISIP 5 x 000 TI UIT IM ulu OT 2 9 ed 3 4ejjng ysom 1 005 gn amp ojgissod sp 3upypuJ4odns J91DM 44 SONY r 02 pup T d 4ejjng 2 USDA YNG yonw SD anoway 6 x 000 9T uiw or spNq 1 02 spNq Ly ULUS Q ir or e4n3xiu uonopeu asONG iri COT dE Bulx 340A Ag je xlui 9
31. lus illustra Ready To Go RT PCR Beads illustra Ready To Go RT PCR Beads illustra Ready To Go RT PCR Beads DNA Polymerization Mix 20 mM each dATP dCTP dGTP dTTP For Protein Urea 1 D or 2 D Electrophoresis Thiourea CHAPS Tris 2 D CLEAN UP KIT 2 D OUANT KIT ECL Western Blotting System 53 Product Code E 1182 E 1182 27 9266 01 27 9267 01 27 9259 01 27 2094 01 17 1319 01 RPN6301 17 1314 01 17 1321 01 80 6484 51 80 6483 56 RPN2108 Pack Size 100 reactions 100 reactions 0 5 ml 100 rxn tubes 0 2 ml 96 rxn tube 0 2 ml 96 rxn h tube 100 umol 500g 100 g 1g 500 g 50 samples 500 assays 1 kit Application For Protein 1 D or 2 D Electrophoresis Product ECL Plex Western Blotting Combination Pack Cy3 Cy5 Hybond ECL Deep Purple Total Protein Stain Full range Rainbow Molecular Weight Markers 54 Product Code RPN998 RPN6305 RPNSOOE Pack Size 1 kit 5 ml 250 ul 7 References 1 Aljanabi S M amp Martinez Nucl Acids Res 25 4692 4693 1997 2 Vogelstein B S Gillespie D Proc Natl Acad Sci USA 76 615 1979 3 Sambrook J amp Russell D W Molecular Cloning A Laboratory Manual chapter 6 2001 55 GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Hea
32. membrane No water drops should stick to the walls of the columns The membrane has to be wetted completely e Ionic strength and pH influence A gt absorption as well as ratio A A gt go thus for absorption measurement use 10 mM Tris HCl pH 7 as diluent and when setting the baseline e Sample material not stored properly Whenever possible use fresh material If this is not possible flash freeze the samples in liguid nitrogen or treat with a stabilizing agent Samples should always be kept at 80 C Never allow tissues to thaw before addition of Lysis buffer type 15 Perform disruption of samples in liguid nitrogen when using mortar and pestle 46 Problem Poor RNA guality or yield continued Possible cause Suggestions Sample material e Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and homogenization e Too much starting material may lead to column clogging or reduced RNA quality or yield RNA quality and yield problems relating to too much sample material may be addressed by decreasing the amount of starting material 47 Problem Clogged during RNA binding Possible cause Sample material incompletely homogenized Suggestions e Centrifuge crude lysate at 11 000 x g to pellet any insoluble material that may clog the column e Reduce the sample amount increase the time for the centrifugation steps and or increase the volume of Lysis buffe
33. nd its contents Please check that no residual flowthrough remains in the column outlet If any remains empty the Collection tube put the column back repeat Step 8b 1min 11 000 x g c Transfer the column to a fresh RNase free 1 5 ml microcentrifuge tube user supplied 9 RNA elution a Add 100 ul Elution buffer type 9 m to the 100 yl Elution center of the column buffer type 9 50 ul or less of Elution buffer type 9 can be used for achieving more concentrated RNA elution b Spin for 1 minute at 11 000 x g to collect the purified total RNA as the flowthrough in the ar Collection tube 1min 11 000 x g 32 c Discard the column and store purified RNA at 80 C Purified RNA is stable for up to 12 months Proceed to Step 10 w Total protein isolqtion 10 Protein precipitation a Transfer the entire flowthrough approximately 600 ul from Step d to a new 1 5 ml microcentrifuge tube Flowthrough containing total protein can be stored at 80 C for up to 6 months b Add 600 ul of Protein precipitation buffer type 1m c Mix vigorously and incubate for 5 minutes at room temperature to precipitate the proteins d Spin for 10 minutes at full speed minimum 1min 16 000 x g 16 000 x g e Carefully remove as much supernatant as possible by pipetting or decanting 11 Protein wash a Add 1 ml of distilled water to protein pellet user suppliedl b Actively disperse p
34. ng accurate analysis of differences in protein abundance between samples It is possible to separate up to three different samples within the same 2 D gel The technology is based on the specific properties of spectrally resolvable dyes CyDye DIGE Fluor dyes As a consequence identical proteins labeled with each of the CyDye DIGE Fluor dyes will migrate to the same position on a 2 D gel This ability to separate more than one sample on a single gel permits the inclusion of up to two samples and an internal standard internal reference in every gel The internal standard is prepared by mixing together egual amounts of each sample in the experiment and including this mixture on each gel 2 D DIGE combines confidence with reproducibility to give dependable results to detect and analyze differences or changes in protein levels expression between complex protein samples To avoid modification of proteins never heat a sample after adding urea If the sample contains urea the solution temperature must not exceed 37 C Elevated temperatures cause urea to hydrolyze to isocyanate which modifies proteins by carbamylation resulting in artifactual charge trains The 2 D Ouant Kit supplied by GE Healthcare should be used to determine protein concentration for protein resuspended in 2 D DIGE buffer Please note 2 D DIGE buffer is not compatible with BCA or Bradford assays Protein resuspended in 2 D DIGE buffer can be also used for SDS
35. ng more concentrated RNA elution f Spin for 1 minute at 11 000 x g to collect the purified total RNA as the flowthrough in the Collection tube 41 6 7 RPM to RCF calculation The appropriate centrifugation speed for a specific rotor can be calculated from the following formula RPM 1000 x RCF 1 12r where RCF relative centrifugal force r radius in mm measured from the center of the spindle to the bottom of the rotor bucket and RPM z revolutions per min e g if an RCF of 735 x g is reguired using a rotor with a radius of 73 mm the corresponding RPM would be 3000 42 6 8 Troubleshooting guide This guide may be helpful in the first instance However if problems persist or for further information please contact GE Healthcare technical services http www gelifesciences com illustra Problem Low genomic DNA yield Possible cause Suggestions Homogenization of e Use of rotor stator homogenizer is tissue incomplete recommended e For difficult to lyse tissues such as mouse or rat tails crushing the animal tissue in liguid nitrogen is recommended prior to proceeding with DNA extraction Tissue sample old or e For best results use fresh tissue samples subjected to repeat freeze thavv cycles Incorrect volume of e Follow the protocol carefully The volume Lysis buffer type 15 of Lysis buffer type 15 is critical used Column clogged due to Do not use more than 20 mg tissue as overloading starting mater
36. ntain the integrity of the RNA and maximize proteome representation The entire procedure can be completed in as little as 45 minutes to yield genomic DNA total RNA and denatured total protein which are compatible with most molecular biology applications These are summarized in Table 1 Table 1 Analyte Downstream applications Genomic DNA Restriction enzyme digestion PCR sequencing array CGH Total RNA RT PCR cDNA synthesis Expression array Total Protein SDS PAGE Western Blotting 2 D DIGE LCMS DNA RNA and protein have been successfully isolated from a range of tissue types such as liver kidney spleen brain lung and intestine and the cell lines HeLa NIH 3T3 CHO K1 and HEK 293 Tissue amounts ranging from 1 20 mg can be used for each preparation Cells amounts ranging from 0 3 5 million cells can be used for each preparation 10 Although the kit is designed to isolate DNA RNA and Protein users may select to only isolate 2 out of 3 analytes when following the protocol As shown in Fig 1 the DNA purification step does not affect RNA and protein isolation RNA purification step does not affect protein isolation RNA in the flowthrough is stable for about 30 minutes at room temperature or 60 minutes on ice depending on sample types Users may purify RNA immediately at this point by following Step 6 before returning to Step 3 for DNA purification to prevent potential RNA degradation DNA bound to the mini column is st
37. r 3 5 minutes e Proceed immediately to Step 2 or store at 80 C for up to 6 months qi Homogenized sample in Lysis buffer type 15 containing 2 Mercaptoethanol may be stored at 80 C for up to 6 months 1 ii Tissue homogenization amp lysis using a mortar amp pestle and a needle amp syringe a Dispense sufficient liguid nitrogen to cover the bottom of a mortar Immediately place tissue 1 20 mg in liguid nitrogen and grind the sample to a fine powder in the presence of liquld nitrogen b Allovv the liguid nitrogen to evaporate but do not allow the tissue to thaw O Add 350 ul of Lysis buffer type 15 containing 2 Mercaptoethanol and grind further with the pestle until no visible tissue pieces are present Pass lysate at least 5 times through a DNase amp RNase free 20 gauge needle fitted with 1 ml sterile syringe D Proceed immediately to Step 2 or store at 80 C for up to 6 months i Homogenized sample in Lysis buffer type 15 containing 2 Mercaptoethanol may be stored at 80 C for up to 6 months 27 1 iii Cell homogenization amp lysis a Pellet up to 5 x 10 cultured cells in a 1 5 ml microcentrifuge tube by centrifugation for 1 minute at 11 000 x g b Completely remove the supernatant by aspiration c Wash the pellet with 500 ul PBS Spin for 1 minute at 11 000 x g Completely remove the supernatant by aspiration el pellet can be stored at 80 C for upto
38. r type 15 Note only load 350 ul of crude lysate onto the 1st mini column e f clogging still occurs take the remaining lysate off the mini column discard it and proceed with the wash step with Wash buffer type 6 Problem Contamination of RNA with genomic DNA Possible cause Too much starting material tissue or cell used DNase not active DNase solution not properly applied Suggestions e Reduce quantity of cells or tissue used e Reconstitute and store lyophilized DNase according to instructions given in section 5 1 e Pipet DNase I solution directly onto the center of the silica membrane 48 Problem Contamination of RNA with genomic DNA Possible cause DNA detection system too sensitive Suggestions e The amount of DNA contamination is significantly reduced by binding DNA in crude lysate to the 1st mini column e On column DNase digestion will remove DNA that goes onto 2nd mini column However residual DNA may still be present due the amount and nature of the starting material Therefore in very sensitive applications it might be possible to detect DNA The potential for DNA contamination detection by PCR increases with e the number of DNA copies per preparation single copy target lt plastidial mitochondrial target lt plasmid transfected into cells e decreasing PCR amplicon size use larger PCR targets e g gt 500 bp or intron spanning primers if possible 49 Problem
39. rotein pellet by pipetting up and down at least 5 times c Spin for 1 minute at full speed minimum 16 000 x gl Position tubes vvithin the microcentrifuge vvith cap hinge facing outward Protein pellet will then form at the bottom of the tube directly beneath the cap hinge 1min16000xg 33 RS A second water wash may be repeated if further removal of residual salt in protein pellet is needed e g 2 D DIGE or LCMS experiments d Carefully remove as much supernatant as possible by pippeting or decanting 12 Protein resuspension a Add 50 500 ul of 2 D DIGE buffer see Page 24 Table 5 for preparation instruction 7 b Incubate for 5 minutes at room temperature Eg The 2 D Quant Kit supplied by GE Healthcare should be used to determine protein concentration Please note 2 D DIGE buffer is not compatible with BCA or Bradford assays Alternatively 7 M Urea can be used for protein resuspension and further diluted to less than 5 M to be compatible with BCA or Bradford assays E amp Protein resuspended in 2 D DIGE buffer can be used for SDS PAGE by mixing with 1 volume of 2090 SDS Add appropriate sample loading buffer i e 1 volume of 2x Laemmli buffer and incubating at 70 C for 10 minutes before gel loading 34 6 Appendices 6 1 Tissue homogenization considerations The complete disruption and homogenization of animal tissue is critical Following homogenization the tissue should be uniformly suspen
40. s at http www gelifesciences com GE Healthcare supplies a wide range of buffer types across the illustra nucleic acid and TRAP protein purification range The composition of each buffer has been optimized for each application and may vary between kits Care must be taken to only use the buffers supplied in the particular kit you are using and not use the buffers supplied in other illustra kits e g the Lysis buffer type 15 supplied in the illustra triplePrep Kit is not the same as Lysis buffers type 1 amp 4 supplied in the illustra tissue amp cells genomicPrep Mini Spin Kit In order to avoid confusion and the accidental switching of buffers between kits a numbering system has been adopted that relates to the entire range of buffers available in the illustra purification range For example there are currently 15 Lysis buffers in the illustra range 6 Wash buffers and 9 Elution buffers denoted by Lysis buffer type 1 15 Wash buffer type 1 6 and Elution buffer type 1 9 respectively Please ensure you use the correct type of Lysis Wash and Elution buffer for your purification 3 2 Materials to be supplied by user Chemicals e 2 Mercaptoethanol 14 3 M or 598906 e Absolute Ethanol molecular biology grade e Nuclease free Water molecular biology grade e Absolute Acetone molecular biology grade e 2 D DIGE buffer for easy protein re suspension 7 M Urea 2M Thiourea 4 v v CHAPS 30 mM Tris HCl pH 8 5 Chemicals in 2 D
41. sers may purify RNA immediately at this point by following Step 6 before returning to Step 3 for DNA purification to prevent potential RNA degradation Experienced users may also simultaneously purify DNA and RNA by performing Steps 3 and 6 4 and 8 5 and 9 at the same time when DNase treatment is not needed 29 FW Please check that no residual flowthrough 3 DNA wash 1 a Add 500 ul of Lysis buffer type 15 i 500 il Lysis buff without 2 mercaptoethanol to the column men type 15 b Spin for 1 minute at 11 000 x g Discard the flowthrough 1 min 11 000 x g c Place the column back into the same Collection tube 4 DNA wash 2 a Add 500 ul of Wash buffer type 6 m to the column 500 ul Wash buffer type 6 b Spin for 1 minute at 11 000 x g Discard the Collection tube and its contents i 1 min 11 000 x g remains in the column outlet If any remains empty the Collection tube put the column back repeat Step 4b c Iransfer the column to a fresh DNase free 1 5 ml microcentrifuge tube user supplied 5 DNA elution a Add 100 ul of Elution buffer type 5 m to the center ofthe column b Spin for 1 minute at 11 000 x g to collect the purified DNA as the flowthrough in the 1 5 ml microcentrifuge tube 1min 11000xg TI LAJ 30 c Discard the column and store purified DNA at 20 C or 80 C If not subjected to repeated freeze thaw cycles DNA is stable for 12 months
42. y hazardous Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products Suitable protective clothing such as laboratory overalls safety glasses and gloves should be worn Care should be taken to avoid contact with skin or eyes if contact should occur wash immediately with water see Material Safety Data Sheet and or Safety Statements for specific recommendations Warning This protocol requires the use of Ethanol and Acetone Lysis buffer type 15 and Protein precipitation buffer type 1 are harmful if ingested inhaled or absorbed through the skin and can cause nervous system disturbances severe irritation and burning They are destructive to the eyes skin and mucous membranes of the upper respiratory tract Gloves should always be worn when handling these solutions Use of this product with cells tissue or tissue products should be considered biohazardous Follow appropriate safety procedures while using this kit and when handling DNA RNA and protein isolated from these sources Waste effluents from this kit should be decontaminated with bleach or by a detergent based method Decontamination with bleach may be reactive resulting in foam and emission of ammonia gas and should be carried out in an exhaust hood Consult local safety regulations for disposal of all waste 2 2 Storage Store lyophilized RNase free DNase I at
43. y UV spectrophotometer and through comparison with a known standard by agarose gel electrophoresis The reliable range of UV spectrophotometric data should be determined for individual spectrophotometers Generally for spectrophotometers with a 1 cm path length A readings should lie between 0 1 and 1 0 and appropriate dilutions 5 to 50 ng ul should be analyzed For NanoVue spectrophotometers absorbance readings between 1 and 10 provide more reliable results The UV spectrophotometric ratios A A provide information regarding the purity of DNA A purity ratio of 1 7 to 1 9 indicates that DNA is pure for all standard molecular biology applications If the ratio is lower than 1 7 the purified DNA might contain some protein impurities Similarly if the ratio is higher than 1 9 the DNA might contain some RNA impurities 1 OD unit A is equivalent to approximately 50 ug ml double stranded DNA Yield Az x 50 ug ml x 0 1 ml the total ug of purified genomic DNA in the sample 38 6 4 RNA handling and auantification RNA should be treated with care because RNA is very sensitive to trace contaminations of RNases often found on general labware fingers and dust It is necessary to create an RNase free working environment Wear gloves at all times during the preparation Change gloves freguently Use of sterile disposable polypropylene tubes is recommended Keep tubes closed whenever possible during the preparation
44. zen cell pellets should not be thawed but processed immediately in Lysis buffer type 15 containing 2 Mercaptoethanol Protocols are provided for use of a rotor stator homogenizer or a mortar and pestle combined with a needle and syringe Either protocol may be followed when processing tissue samples A separate protocol is provided when processing cultured cells If using any instrument for tissue disruption and homogenization ensure that you are familiar with the operating procedure by referring to its user manual and handbook Some instruments may require use of greater than 350 ul Lysis buffer type 15 Please use the volume recommended by your instrument and adjust sample input accordingly Load only 350 ul of crude lysate to the 1st mini column orange o ring 1 i Tissue homogenization amp lysis using a rotor stator homogenizer a Select proper sized generator and suitable sized tube for homogenization of a small volume of sample i e 350 ul or more Add 350 ul of Lysis buffer type 15 containing 2 Mercaptoethanol to the tube O Add tissue sample 1 20 mg d Homogenize the tissue according to instrument s user manual Visually inspect the prepared homogenate and ensure thorough homogenization No tissue pieces should be visible WN Take care to keep the rotor tip submerged in order to avoid excess foaming If foaming 26 occurs during homogenization let the homogenate stand at room temperatures fo
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