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DNA, RNA, and protein purification - MACHEREY

1. For the primary determination of protein concentration of the sample measure different amounts for example 2 uL 10 uL and 50 uL This assures that at least one value of the three amounts tested falls within the range of the calibration curve Further for a first rough estimation of expected protein yield also consider Table 4 in section 2 2 and Table 5 in section 2 4 Materials TCA 60 Trichloracetic acid not supplied Protein Solving Buffer with reducing agent PSB TCEP see note below BSA Bovine Serum Albumin not supplied Multititer plate not supplied Note The volume of PSB TCEP provided with the kit might not be sufficient to quantify all isolated protein samples Additional PSB TCEP can either be ordered separately see ordering information or easily be prepared see composition of PSB TCEP below MACHEREY NAGEL 06 2014 Rev 05 35 Total DNA RNA and protein isolation Composition of PSB TCEP 125 mM BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane 2 SDS sodium dodecyl sulphate 50 mM TCEP Tris 2 carboxyethyl phosphine Hydrochloride 20 glycerol 0 01 bromophenol blue pH 6 8 Note The composition of the previously used Protein Loading Buffer PLB has been improved and is now called Protein Solving Buffer PSB reduced concentration of SDS and bromophenol blue The change in composition has increased the compatibility with protein quantification methods see above
2. Some sample materials may contain DNase traces which are not sufficiently washed away by the standard procedure Perform a wash step of the column with Buffer RA2 after loading the lysate onto the column and before starting the washing steps with DNA Wash solution add 500 uL Buffer RA2 onto the column centrifuge 1 min at 11 000 x g and continue with DNA Wash washing steps Divalent cations Suboptimal Eluted DNA contains small amounts of divalent cations If performance the downstream application comprises for example 50 of DNA in DNA eluate of the final reaction volume the divalent cations downstream introduced into the reaction by the DNA eluate may alter the application performance Decrease the divalent cation concentration of the reaction by 1 5 mM for compensation Sample amount to large Low DNA Depending on the type of sample and its DNA content DNA yield for yield may not increase proportional to increased sample large sample amount Sample amounts larger than for example 5 mg tissue amounts or 10 cultured cells may yield less DNA than smaller sample amounts Use smaller sample to ensure good correlation between sample amount and DNA yield 38 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Problem Possible cause and suggestions RNase contamination RNA is Create an RNase free working environment Wear degraded gloves during all steps of the procedure Change gloves no
3. DNA RNA and protein purification User manual NucleoSpin TriPrep June 2014 Rev 05 MACHEREY NAGEL www mn net com Total DNA RNA and protein isolation Protocol at a glance Rev 05 NucleoSpin TriPrep 1 Homogenize sample e Up to 30 mg 2 Lyse sample T 350 uL RP1 3 5 uL B mercaptoethanol 7 or comparable reducing agent 3 Filtrate lysate El e 1 min 11 000 x g I 4 AdjustDNAand RNA F binding conditions 350 uL ethanol 70 Y 5 Bind DNA and RNA Load sample 30 s 11 000 x g DNA and RNA Purification both bound to the silica membrane gt g Protein Purification protein in the column flow through 6 Wash silica membrane 1 and 2 wash each 500 uL DNA Wash 1 min 11 000 x g 7 Dry mem brane RT 3 min with open lid 8 Elute DNA 100 uL DNA Elute Incubate 1 min 1 min 11 000 x g 9 Digest residual DNA 10 Wash and dry silica membrane 95 uL DNase reaction mixture RT 15 min 1 wash 200 uL RA2 2 wash 600 uL RA3 30 s 11 000 x g 3 wash 250 uL RA3 2 min 11 000 x g 11 Elute highly pure RNA MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com 60 uL H O RNase free 1 min 11 000 x g 12 Precipitate 10 700 uL protein
4. No protein precipitate pellet visible Most commonly used protein quantification systems are incompatible with concentrations of SDS and or reducing agents present in Protein Solving Buffer Use a suitable quantification method as described in section 6 1 e If an other protein dissolution buffer than PSB or PSB TCEP was used for dissolving the protein pellet ensure compatibility of your buffer and quantification method of choice A small sample amount was used and or a small volume of column flow through was used for precipitation Formation of a visible protein pellet is not required for sufficient protein recovery Even invisible protein pellets commonly yield enough protein for SDS PAGE and Western Blot analysis PSB TCEP turbid or partially solidified PSB TCEP may form a precipitate at temperatures below 18 C Warm up 25 C to dissolve turbidity completely No low protein yield Protein was resolubilized in water Due to the strongly denatured form of the protein the solubility in water is significantly reduced Use PSB TCEP for protein solubilization 42 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation 6 3 References The following publications show the general usefulness of the parallel extraction of DNA RNA and protein from small and precious samples Coombs LM Pigott D Proctor A Eydmann M Denner J and Knowles MA 1990 Simultaneous isolation o
5. N VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects
6. Protocols DNA RNA and protein purification from cultured cells and tissue Joint protocol steps for DNA RNA and protein purification DNA purification steps 1 8 RNA purification steps 1 11 Protein purification steps 1 5 and 12 15 Before starting the preparation Check if Buffer DNA Wash Wash Buffer RA3 rDNase and Reducing Agent TCEP were prepared according to section 3 Homogenize sample Disrupt up to 30 mg of human animal tissue or up to 100 mg of plant tissue for homogenization methods see section 2 3 Up to 5 x 10 eukaryotic cultured cells are collected by centrifugation and lysed by addition of Buffer RP1 directly Lyse sample Add 350 uL Buffer RP1 and 3 5 pL B mercaptoethanol B ME to the cell pellet or to ground tissue and vortex vigorously Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RP1 e g ad 7 14 uL of a 500 mM DTT or TCEP solution Disrupt sample 350 pL RP1 ji 3 5 uL B ME MACHEREY NAGEL 06 2014 Rev 05 21 NucleoSpin TriPrep Filtrate lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter Place NucleoSpin Filter violet ring in a Collection Tube 2 mL apply the mixture and centrifuge for 1 min at 11 000 x g The lysate may be passed alternatively 5 times through a 0 9 mm needle 20 gauge fitted to
7. Too much cell material used Reduce quantity of cells or tissue used 40 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Problem Possible cause and suggestions Contamination of RNA with genomic DNA continued DNA detection system too sensitive The amount of DNA contamination is significantly reduced during the on column rDNase digestion Anyhow we can not guarantee that the purified RNA is 100 free of DNA In very sensitive applications it might be possible to detect DNA The NucleoSpin TriPrep system is checked by the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection of a 1 kb fragment in a 30 cycle reaction Generally no PCR fragment is obtained if the rDNase is applied however a strong PCR fragment is obtained if rDNase is omitted The probability of DNA detection with PCR increases with the number of DNA copies per preparation single copy target lt plastidial mitochondrial target lt plasmid transfected into cells decreasing PCR amplicon size Contamination of RNA with genomic DNA continued Use larger PCR targets e g gt 500 bp or intron spanning primers if possible Use support protocol 5 3 for subsequent rDNase diges tion in solution Suboptimal performance of RNA in downstream experiments Carry over of ethanol or salt Do not let the flow t
8. USA IS PROHIBITED FOR PATENT REASONS 44 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation 6 5 Product use restriction warranty NucleoSpin TriPrep kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR
9. arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 06 2014 Rev 05 45 Total DNA RNA and protein isolation components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL publi
10. DNA Elute directly onto the membrane ee and incubate for 1 min Elute DNA by centrifugation for ue 1 min at 11 000 x g Incubate The temperature of the DNA Elute solution shall not exceed 1 min 30 C otherwise RNA will partly elute with the DNA Elute solution DNA Elute solution may stay for 1 min up to 15 min 1 min on the column before DNA is eluted A 1 5 min incubation 11 000 x g time is recommended Eluted DNA is immediately ready for downstream applications without further purification Further steps for RNA purification steps 9 11 9 Digest residual DNA on column Prepare rDNase reaction mixture in a sterile microcentrifuge tube not provided For each isolation add 10 uL reconstituted rDNase also see section 3 to 95 pL 90 uL Reaction Buffer for rDNase Mix by flicking the rDNase tube reaction A mixture Apply 95 pL rDNase reaction mixture directly onto the center of the silica membrane of the column Incubate at RT room temperature for 15 min 15 min Do not centrifuge directly proceed with step 10 Depending on sample type and amount approximatively 50 90 of the DNA is eluted in the DNA elution step Residual DNA on the column is digested on column with rDNase 24 MACHEREY NAGEL 06 2014 Rev 05 NucleoSpin TriPrep 10 Wash and dry silica membrane Add 200 uL Buffer RA2 to the NucleoSpin TriPrep Column Centrifuge for 30s at 11 000 x g Place the NucleoSpin TriPrep Column into a new Collection Tube 2 m
11. For details on the composition of previous Protein Loading Buffer PLB contact our technical service Method Prepare a BSA stock solution with 40 mg mL BSA in H O Prepare a BSA dilution series Tube Add PSB Add BSA solution Resulting BSA Resulting BSA to tube to tube concentration in 20 pL 1 97 5 uL 2 5 uL BSA stock 1 ug uL 20 ug solution 40 mg mL 2 50 uL 50 uL from tube 1 0 5 ug uL 10 ug 3 50 uL 50 uL from tube 2 0 25 ug uL 5 ug 4 50 uL 50 uL from tube 3 0 125 ug uL 2 5 ug 5 50 uL 50 uL from tube 4 0 063 ug uL 1 25 ug 6 50 uL 50 uL from tube 5 0 031 ug L 0 625 ug 7 50 uL _ 0 ug uL 0 ug Make sure that the protein concentration of your sample lies within the range of the largest 1 and smallest 6 value of the calibration curve in order to obtain valid measurements Do not extrapolate beyond this range The prepared BSA dilution series is sufficient for subsequent determination of two calibration curves 36 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation 1 Add 20 uL of each dilution series sample 1 7 in microtiter plate wells 2 Add 20 uL of samples protein dissolved in PSB TCEP with unknown protein concentration to further wells alternatively 1 60 uL 3 Add 40 uL PSB TCEP to each well Final volume 60 uL alternatively add 0 59 uL if other volumes than 20 uL of sample are used in step 2 Add 40 uL TCA 60 to each well Mix until complete
12. MACHEREY NAGEL 06 2014 Rev 05 31 Total DNA RNA and protein isolation 4 Bio Rad DC Protein Assay Dilute the protein sample 1 10 with water to enable compatibility 5 Bio Rad RC DC Protein Assay Dilute the protein sample 1 5 with water to enable compatibility 6 Serva ProtaQuant Assay Kit According to manufacturer s instructions this assay should be compatible with PSB PSB TCEP and Laemmli buffer samples 7 G Biosciences SPN Protein Assay According to manufacturer s instructions this assay should be compatible with PSB PSB TCEP and Laemmli buffer samples 8 Bio Rad Protein Assay Bradford This method has a very low tolerance towards SDS 0 1 SDS for the Standard Assay Procedure Therefore PSB PSB TCEP Laemmli buffer samples have to be diluted considerably with water to reduce interference After dilution of the sample 1 20 with water protein can be quantified with the standard assay procedure The microassay procedure however is not compatible with such samples even after 1 50 dilution of the sample with water Compatibility of protein quantification methods with PSB and PSB TCEP samples Acceptable Protein concentration Protein Input sample volume re protein quantification range tificat g PSB TCEP en BI F ORT amount per undiluted PSB TCEP assay sample assay sample Protein 0 03 1 ug uL tificati Quan mca ion 20 uL standard standard 1 Assay highly 1 6
13. RNA frequently Use of sterile disposable polypropylene tubes is btained recommended Keep tubes closed whenever possible during SANS the preparation Glassware should be oven baked for at least 2 hours at 250 C before use Reagents not applied or restored properly Reagents not properly restored Add the indicated volume of RNase free water to rDNase vial and 96 ethanol to Buffer RA3 Concentrate and mix Reconstitute and store ly ophilized rDNase according to instructions given in section 3 Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added No ethanol has been added after lysis Binding of RNA to the silica membrane is only effective in the presence of ethanol Kit storage e Reconstitute and store lyophilized rDNase according to Poor RNA instructions given in section 3 quality or yield Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and pH influence Azs absorption as well as ratio Azeo A280 e For adsorption measurement use 5mM Tris pH 8 5 as diluent Please see also Manchester KL 1995 Value of Azeo A go ratios for measure ment of purity of nucleic acids Biotechniques 19 208 209 Wilfinger WW Mackey K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assess ment
14. analysis Binding capacity of DNA lt 10 ug strongly depending on RNA amount bound to the membrane MACHEREY NAGEL 06 2014 Rev 05 7 Total DNA RNA and Protein Isolation DNA characteristics Isolated DNA is of high molecular weight and typically exceeds 20 kb DNA is commonly stable even at 37 C for 2 h with or without addition of a typical restriction enzyme buffer showing the absence of DNases DNA is digestable with restriction enzymes and suitable for PCR RNA characteristics The NucleoSpin TriPrep kit allows purification of pure RNA with an Agsgo Avgo ratio generally exceeding 1 9 measured in TE buffer pH 7 5 The isolated RNA is ready to use for applications like reverse transcriptase PCR RT PCR primer extension or RNase protection assays RNA of high integrity can be isolated with NucleoSpin TriPrep kit RIN RNA Integrity Number of RNA isolated from fresh high quality sample material e g eukaryotic cells or fresh mouse liver generally exceeds 9 0 However RNA integrity strongly depends on the sample quality RNA integrity was examined using the Agilent 2100 Bioanalyzer in conjunction with the RNA 6000 Nano or Pico assay RNA prepared with NucleoSpin TriPrep is generally free of residual DNA However minute traces of DNA may remain if large amounts of material rich in nucleic acids are used If the isolated RNA will be used as template in a RT PCR reaction we recommend using lower quan
15. approx 1 mm PSB is recommended TCEP is a powerful multi purpose and odourless reducing agent It is non volatile and unlike commonly used reducing agents like DTT and B mercaptoehanol resistant to air oxidation TCEP reduces disulfide bonds as effectively as dithiothreitol DTT TCEP reduces even most stable water soluble alkyl disulfides selectively and completely over a wide pH range Solubilization of TCEP in PSB according to the instruction results in a PSB TCEP solution with a concentration of 50 mM TCEP see section 6 1 for composition This provides sufficient molar excess to reduce peptide and protein disulfide bonds effectively within a few minutes in a range up to a protein concentration of approximately 1 ug uL 2 5 Elution procedures for DNA Elution of DNA is carried out under selective conditions to make sure that only DNA is eluted while RNA is still bound to the membrane The DNA washing solution DNA Wash and the DNA elution buffer DNA Elute are finely tuned to achieve this Therefore the DNA elution volume should only be altered moderately in the range of 60 150 UL Furthermore the temperature of the DNA Elute solution shall not exceed 30 C otherwise RNA will partly elute with the DNA Elute solution DNA Elute solution may stay for 1 min up to 15 min on the column a 1 5 min incubation time is recommended Eluted DNA is immediately ready for downstream applications without further purification 2 6 Elution proce
16. flow through 1 vol PP RT 10 min ED 5 min 11 000 x g 13 Wash protein 500 uL ethanol 50 pellet 14 Dry protein RT 5 10 min pellet 15 Prepare 20 100 uL protein PSB TCEP sample 3 min 95 98 C i l ED 1min 11 000 x g Total DNA RNA and protein isolation Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 5 1 3 About this user manual 5 2 Product description 6 2 1 The basic principle 2 2 Kit specifications 2 3 Handling preparation and storage of starting materials 12 2 4 Guideline for appropriate sample amount precipitation volume and resolubilization volume for protein isolation 14 2 5 Elution procedures for DNA 15 2 6 Elution procedures for RNA 15 3 Storage conditions and preparation of working solutions 16 4 Safety instructions 19 5 Protocols 21 5 1 DNA RNA and protein purification from cultured cells and tissue 21 5 2 Total RNA preparation from RNA ater treated samples 28 5 3 rDNase digestion in solution 29 6 Appendix 31 6 1 Protein quantification 31 6 2 Troubleshooting 38 6 3 References 43 6 4 Ordering information 44 6 5 Product use restriction warranty 45 MACHEREY NAGEL 06 2014 Rev 05 3 Total DNA RNA and protein isolation 1 Components 1 1 Kit contents NucleoSpin TriPrep 10 preps 50 preps 250 preps REF 740966 10 740966 50 740966 250 Lysis Buffer RP1 10 mL 25 mL 125 mL Buffer DNA Wash
17. selectively elutes DNA and keeps RNA quantitatively on the column Eluted DNA is immediately ready for downstream applications without further purification DNA elution prior to RNA elution does neither compromise RNA quantity nor quality RNA isolated with the NucleoSpin TriPrep kit is of identical quality as RNA isolated with the well proven NucleoSpin RNA kit Residual DNA still bound to the silica membrane is removed by an rDNase solution which is directly applied onto the silica membrane during the preparation RNase free rDNase is supplied with the kit Simple washing steps with two different buffers remove salts metabolites and macromolecular cellular components Pure RNA is finally eluted under low ionic strength conditions with RNase free water supplied Protein is isolated from the column flow through Protein is precipitated in denatured form with a special buffer Protein Precipitator PP which effectively precipitates protein After a washing step the protein pellet is dissolved in Protein Solving Buffer PSB containing the odourless reducing agent TCEP The protein can thus readily be applied to SDS PAGE analysis The kit is not recommended for isolation of native proteins 6 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation DNA RNA and protein preparation using NucleoSpin TriPrep kits can be performed at room temperature The DNA and RNA eluates however should be treated with care because R
18. the column membrane Protein is in the flow through Maximal loading capacity of NucleoSpin TriPrep Columns is 750 uL Repeat the procedure if larger volumes are to be processed For DNA and RNA isolation continue with step 6 It is recommended to continue the DNA and RNA isolation protocol first and to perform the protein purification subse quently For protein isolation recover flow through and continue with step 12 The protein containing flow through is stable for several hours at 4 8 C Wash silica membrane Add 500 uL DNA Wash to the NucleoSpin TriPrep Column Centrifuge for 1 min at 11 000 x g Discard flow through and reuse the Collection Tube Add again 500 pL DNA Washto the NucleoSpin TriPrep Column Centrifuge for 1min at 11 000 x g Discard Collection Tube with flow through Chaotropic salt is removed by these washing steps DNA and RNA are still bound to the membrane The membrane is prepared for subsequent DNA elution amp Load sample 30s 11 000 x g 500 uL DNA Wash 1 min 11 000 x g 500 pL DNA Wash 1 min 11 000 x g MACHEREY NAGEL 06 2014 Rev 05 23 NucleoSpin TriPrep Dry membrane Insert the NucleoSpin TriPrep Column into a 1 5 mL microcentrifuge tube not provided Open the lid of the column and let it stand for 3 min This step ensures removal of residual ethanol Elute DNA RT 3 min with open lid Add 100 uL
19. 0 uL optional 0 6 20 ug recommended Kap 0 01 20 ug uL REF 740967 optional Karlsson protocol 0 03 1 ug uL 2 recommended 20 uL standard 0 6 20 ug standard see page 35 for 1 60 uL optional 0 01 20 pg L details optional 25 uL of a 1 Pierce BCA Protein kerni oo 3 Assay Kit reducing P pe 3 125 50 ug 0 625 10 ug uL corresponding to 5 uL Agent compatible patible original sample Method tested in MN laboratory for compatibility with PSB PSB TCEP and Laemmli buffer protein samples 32 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Acceptable Protein concentration Proteln Input sample volume protein quantification range quantification e g PSB TCEP diluted PSB TCEP assay sample amount per undilute assay sample 100 uL of a 1 10 prediluted sample corresponding to 10 uL original sample 20 150 ug 4 Bio Rad DC Protein standard assay or standard Se Assay 5 uLofa 1 10 eg BB prediluted sample micro corresponding to 0 5 uL original sample micro testtube assay 100 uL of a 1 5 prediluted sample correspoding to 20 uL original sample or 20 150 pg 5 Bio Rad RC DC standard assay standard 1 7 5 valu Protein Assay 25 jL of a 1 5 5 0 37 5 ug acs prediluted sample micro corresponding to 5 uL original sample micro testtube assay Serva ProtaQuant 6 20 uL 5 35 0 25 1 75 ug L Assay Kit H Hg Hg H G Bioscience 7 SPN P
20. 12 mL 12 mL 5x12 mL Concentrate Buffer DNA Elute 1 2 mL 12 mL 3x12 mL Wash Buffer RA2 15 mL 15 mL 80 mL Wash Buffer RA3 6 mL 12 mL 3 x 25 mL Concentrate RNase free H O 13 mL 13 mL 60 mL Protein Precipitator PP 9 mL 45 mL 225 mL Protein Solving Buffer PSB 2x1 mL 7 5 mL 40 mL without reducing agent Reducing Agent TCEP 2x 14mg 107 mg 5 x 107 mg Reaction Buffer for rDNase 7mL 7mL 30 mL rDNase RNase free 1 vial size C 1 vial size D 5 vials size D lyophilized NucleoSpin Filters 10 50 250 violet rings NucleoSpin TriPrep 10 50 250 Columns light blue rings plus Collection Tubes Collection Tubes 2 mL 30 150 750 Collection Tubes 1 5 mL 20 100 500 User manual For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation 1 2 Reagents consumables and equipment to be supplied by user Reagents 50 ethanol to prepare Buffer DNA Wash 70 ethanol to adjust RNA binding conditions 96 100 ethanol to prepare Wash Buffer RA3 Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP Tris 2 carboxyethyl phosphine hydrochloride to supplement lysis buffer Consumables 1 5 mL microcentrifuge tubes for sample lysis and DNA elution Disposable RNase free pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes e Vortex mixer Thermal heating block Equipment for sampl
21. 50 uL 700 uL 35 uL 350 uL 700 uL 35 uL 350 uL 700 uL Buffer PSB used for protein pellet solubilization 100 uL 100 uL 20 uL 100 uL 100 uL 20 uL 100 uL 100 uL 20 uL Protein sample to be analyzed by SDS PAGE with Coomassie stain 10 pL Protein sample to be analyzed by SDS PAGE with silver stain 1 uL Protein sample to be analyzed by Western Blot 1 10 uL Protein pellets with a diameter of up to approximately 1 2 mm in size are ideally suited for subsequent solubilization Protein pellets exceeding volumes of approximately 10 uL should be avoided as large protein pellets are harder to dissolve than small pellets To obtain small protein pellets adapt the volume of column flow through in respect to the amount of sample material Commonly small and even invisible protein pellets yield sufficient protein for SDS PAGE and Western Blot analysis 14 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Solubilization of protein pellets and reduction of protein disulfide bonds The NucleoSpin TriPrep kit provides a protein sample buffer Protein Solving Buffer PSB and the Reducing Agent TCEP The Protein Solving Buffer PSB is similar in composition and function to the buffer commonly known as Laemmli buffer For most applications PSB may be substituted by Laemmli buffer However for applications with large protein pellets
22. CEP Getz EB Xiao M Chakrabarty T Cooke R and Selvin PR 1999 A comparison between the sulfhydryl reductants Tris 2 carboxyethyl phosphine and Dithiothreitol for use in protein biochemistry Analytical Biochemistry 273 73 80 The following publication describes a method for quantification of protein dissolved in sample buffer such as PSB Karlsson JO Ostwald K Kabj rn C and Andersson M 1994 A method for protein assay in Laemmli buffer Analytical Biochemistry 219 144 146 MACHEREY NAGEL 06 2014 Rev 05 43 Total DNA RNA and protein isolation 6 4 Ordering information Product REF Pack of NucleoSpin TriPrep 740966 10 50 250 10 50 250 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 Protein Quantification Assay 74096 7 50 250 50 250 NucleoSpin RNA 740955 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA Midi 740962 20 20 NucleoSpin RNA Clean up 740948 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA DNA Buffer Set 740944 100 Buffer RP1 740934 50 50 mL Buffer RP1 740934 500 500 mL nn Buffer Set 740941 1 set rDNase Set 740963 1 set NucleoSpin Filters 740606 50 Collection Tubes 2 mL 740600 1000 Porablot See price list Blotting Paper See price list Visit www mn net com for more detailed product information DISTRIBUTION AND USE OF NUCLEOSPIN TRIPREP and NUCLEOSPIN RNA DNA BUFFER SET IN THE
23. L Buffer RA2 will inactivate the rDNase Add 600 uL Buffer RA3 to the NucleoSpin TriPrep Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the NucleoSpin TriPrep Column back into the Collection Tube Add 250yL Buffer RA3 to the NucleoSpin TriPrep Column Centrifuge for 2 min at 11 000 x g to dry the membrane completely Place the NucleoSpin TriPrep Column into an RNase free Collection Tube 1 5 mL supplied If for any reason the liquid level in the Collection Tube has reached the NucleoSpin TriPrep Column after centrifuga tion discard flow through and centrifuge again 11 Elute highly pure RNA Elute the RNA in 60 uL RNase free H O supplied and centrifuge at 11 000 x g for 1 min If higher RNA concentrations are desired elution can be done with 40 uL Overall yield however will decrease when using smaller volumes For further alternative elution procedures see section 2 6 Further steps for protein purification steps 12 15 200 pL RA2 30s 11 000 x g 600 pL RA3 30s 11 000 x g 250 pL RA3 2 min 11 000 x g 60 pL RNase free H O 1 min 11 000 x g Use the NucleoSpin TriPrep Column flow through from step 5 i e the ethanolic lysate which has been passed throught the NucleoSpin TriPrep Column as starting point for protein precipitation MACHEREY NAGEL 06 2014 Rev 05 25 NucleoSpin TriPrep 12 Precipita
24. NA is very sensitive to trace contaminations of RNases often found on general lab ware fingerprints and dust DNA can be stored at 4 C for short term and at 20 C for long term storage To ensure RNA stability keep RNA frozen at 20 C for short term or 70 C for long term storage Recovered protein dissolved in Protein Solving Buffer is unproblematic concerning stability 2 2 Kit specifications NucleoSpin TriPrep kits are recommended for the isolation of total DNA RNA and protein from cultured cells tissue and other biological samples Table 1 Kit specifications at a glance Parameter NucleoSpin TriPrep Format Sample material Mini spin column lt 5 x 10 cultured cells lt 30 mg human animal tissue lt 100 mg plant tissue Total RNA Total DNA Total protein Fragment size gt 200 b lt 30 kbp 15 300 kDa Typical yield lt 70 ug lt 6 ug lt 1200 ug A eo go 1 9 2 1 1 7 1 9 Typical RIN gt 9 _ RNA integrity number Elution volume 40 120 uL 100 uL 10 100 uL RNA and DNA Resolubilization volume protein Preparation time 30 min 6 preps 45 min 6 preps 35 min 6 preps RNA DNA Binding capacity 200 ug 10 ug For samples larger than approx one million cells or 5 mg tissue DNA yield does not increase lineary with the sample amount DNA yield may decrease utilizing five million cells 30 mg tissue or more Typically however DNA yield is still sufficient for PCR
25. a syringe In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to anew 2 mL centrifuge tube not included Important To process higher amounts of cells gt 1 x 10 or tissue gt 10 mg the lysate should first be homogenized using the 0 9mm needle 20 gauge followed by filtration through NucleoSpin Filter Adjust DNA and RNA binding conditions Discard the NucleoSpin Filter and add 350 uL ethanol 70 to the homogenized lysate and mix by pipetting up and down approx 5 times Alternatively transfer flow through into a new 1 5 mL microcentrifuge tube not provided add 350 uL ethanol 70 and mix by vortexing 2 x 5 s After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Make sure to disaggregate any precipitate by mixing and load all of the disaggregated precipitate on the column as described in step 5 Do not centrifuge at this stage in order to avoid sedimentation of any precipitate 1 min 11 000 x g 350 uL 70 EtOH Mix 22 MACHEREY NAGEL 06 2014 Rev 05 NucleoSpin TriPrep Bind DNA and RNA For each preparation take one NucleoSpin TriPrep Column light blue ring placed in a Collection Tube and load the lysate Centrifuge for 30 s at 11 000 x g Place the NucleoSpin TriPrep Column in a new Collection Tube 2 mL RNA and DNA are bound to
26. aemmli buffer recommended Quantification of protein dissolved in alternative protein dissolution buffers 1 SDS or 8 M urea Quantification of protein within the column flow through i e prior to protein precipitation at step 5 of the standard protocol For most reliable results and convenience we recommend the MACHEREY NAGEL Protein Quantification Assay for ordering information see section 6 4 to quantify protein dissolved in PSB PSB TCEP or Laemmli buffer Quantification of protein dissolved in PSB or PSB TCEP The concentation of protein dissolved in PSB PSB TCEP or Laemmli buffer can be determined with several methods Below you find a choice of quantification methods which are compatible with PSB PSB TCEP and Laemmli buffer but not all of the methods show the same sensitivity The following list compares the different sensitivies and gives assistance to find out the most suitable protein quantification assay 1 Protein Quantification Assay highly recommended method This is the most sensitive and convenient method for protein quantification in PSB or PSB TCEB Highly recommended due to sensitivity and high compatibility For ordering information see section 6 4 2 Method adapted from the publication Karlsson et al 1994 For a detailed protocol of the Karlsson method see page 35 3 Pierce BCA Protein Assay Kit reducing agent compatible Dilute the protein sample 1 5 with water to enable compatibility
27. colour change from blue to yellow Incubate for 30 min 3 min at room temperature Measure extinction at 570 nm N 9 9 A Determine protein concentration of samples in relation to dilution series E570 3 mm path 0 001 pitty po iy po iis 0 1 1 10 100 BSA amount per well ug Figure 1 BSA standard curve for determination of protein in Protein Solving Buffer PSB Measurement of extinction in the range of 530 700 nm is suitable and will typically result in correlation coefficients of 0 99 concentration of BSA dilution series vs obtained absorption values MACHEREY NAGEL 06 2014 Rev 05 37 Total DNA RNA and protein isolation 6 2 Troubleshooting Problem Possible cause and suggestions DNAis Buffer temperature contaminated DNA elution buffer DNA Elute exceeded 30 C during with RNA application Use DNA Elute with a temperature preferentially of 18 25 C DNA yield Sample material lower than DNA and RNA yield depend very much on sample material RNA yield Ratio of RNA yield to DNA yield may vary from approximately 1 20 DNase contamination DNAelution buffer DNA Elute does not contain divalent cation complexing substances e g EDTA Therefore DNA is not protected against DNases Keep DNA Elute solution clean and avoid any contamination As a precaution keep DNA on DNA degrades ice for short term or at 20 C for long term storage upon storage
28. d be stored at room temperature 18 25 C and are stable up to one year Storage at lower temperatures may cause precipitation of salts Before starting any NucleoSpin TriPrep protocol prepare and consider the following Check that 70 ethanol is available as additional solution to adjust binding conditions in the RP1 lysate Check that 50 ethanol is available as additional solution to wash the protein pellet and to prepate Buffer DNA Wash Check that 96 100 ethanol is available to prepare Wash Buffer RAS The DNA Wash solution is delivered as a concentrate To prepare the final DNA Wash solution add four volumes of 50 ethanol to the DNA Wash Concentrate as indicated in the table below Mark the label of the bottle to indicate that the ethanol is added Keep the bottle tightly close to avoid evaporation of ethanol Due to its composition the DNA Elute solution DNA elution buffer does not inhibit DNases that is DNA Elute does not contain substances e g EDTA to complex divalent cations Therefore be aware not to contaminate DNA Elute with DNases Further due to its composition DNA Elute solution does not inhibit microbial growth Therefore make sure not to contaminate DNA Elute with any source of microbial contamination rDNase RNase free Add indicated volume of RNase free H O see table below to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vials to completely dissolve the rDNase Be care
29. detectable level is challenging and the efficiency of an on column DNA digestion is sometimes not sufficient for downstream applications requiring lowest residual content of DNA A typical example for such a demanding application is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contaminating genomic DNA Especially if high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections the target gene is of a very low expression level the amplicon is relatively small lt 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However stringent RNase control and subsequent repurification of the RNA in order to remove buffer salts DNase and digested DNA are usually required The high quality recombinant RNase free DNase rDNAse in the NucleoSpin TriPrep kits facilitates such a digestion in solution in order to remove even traces of contaminating DNA A Digest DNA reaction setup Add 6 uL Reaction Buffer for rDNase and 0 6 pL rDNase to 60 uL eluted RNA Alternatively premix 100 uL Reaction Buffer for rDNase and 10 uL rDNase and add 1 10 volume to one volume of RNA eluate B Incubate sample Incubate for 10 min at 37 C MACHEREY NAGEL 06 2014 Rev 05 29 NucleoSpin TriPrep Repurify RNA Repurify RNA with a suitable RNA clean up procedure for examp
30. dissolving and denaturation Let sample cool down to room temperature Centrifuge for 1 min at 11 000 x g to pellet residual insolvable material Note Depending on sample amount and nature there might be no visible pellet of insolvable material up to large pellets of different size and structure Do not disturb residual precipitates at this stage Protein will be in the supernatant Do not centrifuge samples at temperatures lt 18 C SDS may precipitate at this temperature Recover supernatant for further analysis See section 6 1 for suitable protein quantification methods Note At this stage samples can be stored at 20 C for several months or at 4 C for several days After storage equilibrate sample to room temperature mix and then centrifuge briefly before withdrawal of sample aliquots Repeated sample denaturing for 3 min at 95 98 C is not necessary Repetitive withdrawal freezing and thawing for at least three times has shown constant sample quality Due to the strong denaturing purification method protein is precipitated in denatured form with reduced solubility in water Therefore resolubilization of the protein pellet in PSB TCEP or in traditional Laemmli buffer is recommended The use of Protein Solving Buffer PSB is not mandatory for dissolving protein Alternatively to PSB PSB TCEP or Laemmli buffer precipitated protein can be dissolved in 1 SDS or 8 M urea Further the protein pellet can be dissolved in urea th
31. dures for RNA It is possible to adapt elution method and elution volume of water to the subsequent application of interest In addition to the standard method described in the individual protocols recovery rate about 70 90 there are several modifications possible High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 of bound nucleic acid will be eluted High yield and high concentration Elute with the standard elution volume and apply the eluate once more onto the column for reelution Eluted RNA should immediately be put on ice and always kept on ice for optimal stability and to avoid RNA degradation by almost omnipresent RNases general lab ware fingerprints dust For short term storage freeze at 20 C for long term storage freeze at 70 C MACHEREY NAGEL 06 2014 Rev 05 15 Total DNA RNA and protein isolation 3 Storage conditions and preparation of working solutions Attention Buffers RP1 and RA2 contain chaotropic salt Wear gloves and goggles CAUTION Buffers RP1 and RA2 contain guanidinium thiocyanate which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Storage conditions Store lyophilized rDNase RNase free at 4 C on arrival stable up to 1 year Store Reducing Agent TCEP at 4 C on arrival All other kit components shoul
32. e disruption and homogenization Personal protection equipment lab coat gloves goggles Additional material is furthermore needed for protein quantification see section 6 1 1 3 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin TriPrep kit is used for the first time Experienced users however may refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 06 2014 Rev 05 5 Total DNA RNA and protein isolation 2 Product description 2 1 The basic principle Introduction Studies of gene expression at the level of transcription and translation by quantification of RNA and protein combined with verification of genomic sequence e g transgene integration site are often hampered by the small sample size and the necessity of different often incompatible techniques for DNA RNA and protein isolation Samples may comprise biopsies tumors tissues transgene organisms and others The NucleoSpin TriPrep kit enables isolation of DNA RNA and protein from diverse sample types DNA RNA and protein ar
33. e isolated without splitting the sample prior to extraction Thus DNA RNA and protein are obtained from one and the same sample and not from three similar portions of one sample This is especially valuable for unique small and precious samples DNA and RNA are eluted separately from the NucleoSpin TriPrep Column with a low salt buffer and water respectively Isolated DNA and RNA are suitable for all common downstream applications Isolated protein is suitable for SDS PAGE Western Blot analysis and quantification DNA RNA and protein isolation One ofthe most important aspects in the isolation of DNA RNA and protein isto prevent their degradation during the isolation procedure Withthe NucleoSpin TriPrep method cells are lysed by incubation in a solution containing large amounts of chaotropic ions This lysis buffer immediately inactivates virtually all enzymes e g DNases RNases proteases and phosphatases which are present in almost all biological materials The buffer dissolves even hardly soluble protein creates appropriate binding conditions which favor adsorption of DNA and RNA to the silica membrane and enables protein to pass the specially treated NucleoSpin TriPrep Column virtually quantitatively Expensive and harmful proteinase inhibitors or inhibitor cocktails are not necessary due to the denaturing properties of the lysis buffer After two special washing steps DNA is eluted with a low salt buffer DNA Elute which
34. f DNA RNA and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiodyanate Analytical Biochemistry 188 338 343 Banerjee S Smallwood A Chambers AE and Nicolaides K 2003 Quantitative recovery of immunoreactive proteins from clinical samples following RNA and DNA isolation BioTechniques 35 3 450 456 Hoemann CD Sun J Chrzanowski V and Buschmann MD 2002 A multivalent assay to detect glycosaminoglycan protein collagen RNA and DNA content in milligam samples of cartilage or hydrogel based repair cartilage Analytical Biochemistry 300 1 10 The following publications cite the use of the NucleoSpin kits for DNA RNA and protein isolation Rodriguez Jim nez FJ Moreno Manzano V Lucas Dominguez R and Sanchez Puelles JM 2008 Hypoxia Causes Down Regulation of Mismatch Repair System and Genomic Instability in Stem Cells Stem Cells May 2008 10 1634 stemcells 2007 1016 Bahn A Hagos Y Reuter S Balen D Brzica H Krick W Burckhardt BC Sabolic and Burckhardt G 2008 Identification of a new urate and high affinity nicotinate transporter human organic anion transporter 10 hOAT10 SLC22A13 J Biol Chem published 14 April 2008 10 1074 jbc M800737200 Weiske J Albring KF and Huber O 2007 The tumor suppressor Fhit acts as a repressor of B catenin transcriptional activity PNAS Dec 2007 104 20344 20349 The following publication decribes the Reducing Agent T
35. ful not to mix rDNase vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at 18 C The frozen working solution is stable for 6 months Do not freeze thaw the aliquots more than three times 16 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Wash Buffer RA3 Add the indicated volume of 96 100 ethanol see table below to Buffer RA3 Concentrate Mark the label of the bottle to indicate that the ethanol is added Store Wash Buffer RA3 at room temperature 18 25 C for up to one year Keep the bottle tightly close to avoid evaporation of ethanol Protein Solving Buffer PSB and Reducing Agent TCEP For SDS PAGE under reducing conditions most common type of SDS PAGE transfer PSB from one vial to one vial of the Reducing Agent TCEP Mix gently to avoid excessive foaming until the reducing agent is dissolved completely this process will require several minutes Protein Solving Buffer containing Reducing Agent TCEP PSB TCEP is stable for several days at room temperature 18 25 C and several months at 4 C For long term storage of PSB TCEP keep at 20 C If SDS PAGE under non reducing conditions is intended consider the following A Omit addition of the Reducing Agent TCEP to Buffer PSB B Omit addition of B mercaptoethanol or other reducing agent to Lysis Buffer RP1 If other reducing agents than TCEP are preferred e g DTT 8B mercaptoethanol a
36. gt 0 98 gt 0 98 Protein gt 0 99 gt 0 99 MACHEREY NAGEL 06 2014 Rev 05 9 Total DNA RNA and protein isolation 1000 100 0 1 DNA RNA protein yield yg Protein RNA DNA 0 01 0 01 0 1 1 Mouse liver amount mg Figure 2 DNA RNA and protein yield from different amounts of mouse liver tissue DNA RNA and protein were isolated from different amounts of mouse liver tissue For tissue amounts exceeding 2 5 mg only a fraction of the protein solution was used for precipitation and quantification total protein yield was calculated DNA RNA and protein were isolated as described in Figure 2 Obtained correlation coefficients between sample amount and DNA RNA and protein yield are shown in Table 3 Table 3 Correlation between sample amount nucleic acid and protein yield 0 08 1 25 mg 0 08 2 5 mg 0 08 5 mg mouse liver mouse liver mouse liver DNA gt 0 99 gt 0 95 gt 0 67 RNA gt 0 98 gt 0 98 gt 0 98 Protein gt 0 99 gt 0 99 gt 0 99 10 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Protein yield Protein yield depends on sample type amount and quality as well as on homogenization efficiency Further the utilized quantification method influences determined protein yield The following values were determined with MACHEREY NAGEL s Protein Quantification Assay see ordering informatio
37. hod is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 3 Bio Rad DC Protein Assay This method is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 4 Bio Rad RC DC Protein Assay This method is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 5 Roti Nanoquant Assay This method is compatible with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 Protein quantification in alternative protein pellet dissolution buffers The use of the PSB or PSB TCEP buffer is not mandatory for solving proteins Precipitated protein protein pellets may be dissolved in alternative solutions such as 1 SDS or 8 M urea or in urea thiourea CHAPS buffers as used for 2 D electrophoresis However depending on the target protein the yield of solubilized protein may be reduced compared to PSB or PSB TCEP Check manufacturers product information to ensure compatibility of your protein quantifiction assay with your alternative protein dissolving solution 34 MACHEREY NAGEL 06 2014 Re
38. hrough touch the column outlet after the second RA3 wash Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Buffer RA3 completely Check if Buffer RA3 has been equilibrated to room temperature before use Washing at lower temperatures lowers efficiency of salt removal by Buffer RAS Store isolated RNA properly Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases general lab ware fingerprints dust will degrade the isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C MACHEREY NAGEL 06 2014 Rev 05 41 Total DNA RNA and protein isolation Problem Possible cause and suggestions Trouble with resolubilization of precipitated Protein pellets exceeding several millimeters in size are hard to dissolve Use smaller volumes of column flow through for protein precipitation in order to obtain small sized pellets Even Be invisible protein pellets commonly yield enough protein for SDS PAGE and Western Blot analysis Protein Protein pellet has not been dried sufficiently and contains residual dissolved in ethanol PSB TCEP Increase drying time or decrease pellet size by precipitating a flows out of smaller volume of column flow through SDS PAGE gel slot immediately after loading Unclear results with commonly used protein quantification systems
39. iourea CHAPS buffers as used for 2 D electrophoresis However depending on the target protein the overal yield of solubilized protein may be reduced compared to PSB or PSB TCEP used as dissolving agent MACHEREY NAGEL 06 2014 Rev 05 27 NucleoSpin TriPrep 5 2 Total RNA preparation from RNAl ater treated samples Before starting the preparation Check if Buffer DNA Wash Wash Buffer RA3 rDNase and Reducing Agent TCEP were prepared according to section 3 1 Prepare sample Remove RNAlater solution Cut an appropriate amount of tissue 2 Lyse sample Add 350 uL Buffer RP1 and 3 5 pL B mercaptoethanol B ME to the sample Disrupt the sample material by using for example rotor stated homogenizers for homogenization methods see section 2 3 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RP1 Proceed with step 3 of the NucleoSpin TriPrep standard protocol section 5 1 28 MACHEREY NAGEL 06 2014 Rev 05 NucleoSpin TriPrep 5 3 rDNase digestion in solution The on column rDNase digestion in the standard protocol is very efficient and results in minimal residual DNA content of the purified RNA This DNA will not be detectable in most downstream applications but there are still certain applications which require even lower contents of residual DNA However removal of DNA to a completely un
40. isruption and that the viscosity of the sample is reduced by homogenization The most commonly used technique for disruption of animal tissues is grinding with a pestle and mortar Grind the sample to a fine powder in presence of liquid No Take care that the sample does not thaw during or after grinding or weighing and add the frozen powder to an appropriate aliquot of Buffer RP1 containing B mercaptoethanol and mix immediately The broken up tissue must then be homogenized with a NucleoSpin Filter or by passing 5 times through a 0 9 mm syringe needle Thawing of undisrupted animal tissue should exclusively be done in presence of Buffer RP1 during simultaneous mechanical disruption for example with a rotor stator homogenizer This ensures that RNA is not degraded by RNases before the preparation starts The spinning rotor disrupts and simultaneously homogenizes the sample by mechanical shearing within seconds up to minutes homogenization time 12 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation depends on sample Take care to keep the rotor tip submerged in order to avoid excess foaming To degenerate evolved foam centrifuge 1 min at 400 x g Select a suitably sized homogenizer 5 7 mm diameter rotors can be used for homogenization in microcentrifuge tubes have to be incubated in lysozyme or lyticase zymolase solutions respectively By this treatment the robust cell walls of these organisms are digested o
41. le by use of the NucleoSpin RNA Clean up or NucleoSpin RNA Clean up XS kit see ordering information or by ethanol precipitation Ethanol precipitation exemplary Add 0 1 volume of 3 M sodium acetate pH 5 2 and 2 5 volumes of 96 100 ethanol to one volume of sample Mix thoroughly Incubate several minutes to several hours at 20 C or 4 C Note Choose long incubation times if the sample contains low RNA concentration Short incubation times are sufficient if the sample contains high RNA concentration Centrifuge for 10 min at max speed Wash RNA pellet with 70 ethanol Dry RNA pellet and resuspend RNA in RNase free H O 30 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation 6 Appendix 6 1 Protein quantification Quantification of protein dissolved in sample buffer such as PSB PSB TCEP or traditional Laemmli buffer is occasionally required prior to SDS PAGE and Western Blot analysis However major protein quantification assays are influenced by or are incompatible with SDS and or reducing agents commonly present in protein sample buffers used for SDS PAGE A protein quantification procedure has to be chosen carefully to ensure appropriate compatibility of the method with the protein dissolution solution The NucleoSpin RNA Protein procedure allows several protein quantification methods at different steps of the procedure Quantification of protein dissolved in PSB PSB TCEP or L
42. n and shall serve as a guideline for expected protein yield It is assumed that the complete sample amount is processed that is the complete lysed sample after ethanol addition is loaded onto the column and the complete 700 uL flow through is subjected to protein precipitation Note that in many cases precipitation of only a portion of the column flow through e g 100 uL is recommended and will yield enough protein in terms of absolute amount and concentration for SDS PAGE and Western Blot analysis As a guideline for appropriate precipitation volumes see section 2 4 Table 4 Typical protein yield Sample type and amount Protein yield Cultured human cells e g HeLa approx 10 cells 50 150 ug Plants e g garden cress approx 100 mg 150 350 ug Animal tissue e g pig liver approx 30 mg 500 1200 ug MACHEREY NAGEL 06 2014 Rev 05 11 Total DNA RNA and protein isolation 2 3 Handling preparation and storage of starting materials RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Therefore it is important that samples are flash frozen in liquid N immediately and stored at 70 C or processed as soon as possible Samples can be stored in Lysis Buffer RP1 after disruption at 70 C for up to one year at 4 C for up to 24 hours or up to several hours at room temperature Frozen samples are s
43. oducts and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 46 MACHEREY NAGEL 06 2014 Rev 05
44. of nucleic acid purity Biotechniques 22 474 481 MACHEREY NAGEL 06 2014 Rev 05 39 Total DNA RNA and protein isolation Problem Possible cause and suggestions Poor RNA quality or yield continued Sample material Sample material was not stored properly Whenever possible use fresh material If this is not possible flash freeze the samples in liquid N Samples should always be kept at 70 C Never allow tissues to thaw before addition of Buffer RP1 Perform disruption of samples in liquid No Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Clogged NucleoSpin Column Poor RNA quality or yield Sample material Too much starting material used Overloading may lead to decreased overall yield Reduce amount of sample material or use larger volume of Buffer RP1 Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Contamination rDNase not active Reconstitute and store Iyophilized rDNase according to instructions given in section 3 rDNase solution not properly applied BERN Pipette rDN lution directly onto the center of the sili genomic DNA ipette rDNase solution directly onto the center of the silica membrane
45. ppropriate amounts should be added to PSB Please consider limited stability of DTT compared to TCEP If PSB TCEP is turbid warm up PSB TCEP to gt 25 C before use until solution is completely clear i e all precipitate is dissolved completely PSB TCEP has a half life of approximately 5 months if stored at 4 C and approximately 7 months if stored at 20 C For 50 and 250 prep kits For better handling PSB TCEP may be transferred into the original PSB vial with screw Cap MACHEREY NAGEL 06 2014 Rev 05 17 Total DNA RNA and protein isolation NucleoSpin TriPrep 10 preps 50 preps 250 preps REF 740966 10 740966 50 740966 250 Buffer 12 mL 12 mL 5x12 mL DNA Wash Add 48 mL Add 48 mL Add 48 mL 50 Concentrate 50 ethanol 50 ethanol ethanol to each vial Wash 6 mL 12 mL 3 x 25 mL Buffer RA3 Add 24 mL Add 48 mL Add 100 mL Concentrate 96 100 ethanol 96 100 ethanol 96 100 ethanol to each vial rDNase 1 vial size C 1 vial size D 5 vials size D RNase free Add 230 uL Add 540 uL Add 540 uL Iyphilized RNase free H O RNase free H O RNase free H O to each vial Reducing 2x 14 mg 107 mg 2x 107 mg Agent TCEP Add 1 mL PSB each Add 7 5 mL PSB Add 7 5 mL PSB each 18 MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation 4 Safety instructions The following components of the NucleoSpin TriPrep kits contain hazardous contents Wear gloves and goggles and follow the safe
46. r at least weakened which is essential for effective cell lysis by Buffer RP1 For microorganisms with extremely resistant cell walls like some Gram positive bacterial strains it may be necessary to optimize the conditions of the treatment with lytic enzymes or the cultivation conditions After lysis homogenization is achieved by using a NucleoSpin Filter or the syringe needle method MACHEREY NAGEL 06 2014 Rev 05 13 Total DNA RNA and protein isolation 2 4 Guideline for appropriate sample amount precipitation volume and resolubilization volume for protein isolation The following Table 5 shall serve as a first guide for choosing appropriate amounts of sample material precipitation volume and resolubilization volume Depending on sample type and downstream application e g Coomassie or silver stain sensitivity of antibody detection system appropriate volumes might deviate from the table below and have to be determined experimentally Table 5 Guideline for appropriate sample amount Cultivated cells Animal tissue Plant tissue Amount of e g garden e g HeLa e g liver cress leaf Sample 10 10 104 30 mg 3 mg 0 3 mg 100 mg 10 mg 1 mg Lysis Buffer RP1 incl reducing 350 uL agent Ethanol 70 350 uL Column flow through to be 35 uL 350 uL 700 uL 35 uL 350 uL 700 uL 35 uL 350 pL 700 uL precipitated Volume of Protein Precipitator PP 35 uL 3
47. resh air and keep at rest in a position com fortable for breathing Bei Einatmen An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sptilen Rinse mouth Mund aussp len If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen IF skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 20 MACHEREY NAGEL 06 2014 Rev 05 NucleoSpin TriPrep
48. rotein 1 10 uL 0 5 10 ug 0 05 10 ug uL Assay Bio Rad Protein 100 uL of 1 20 Assay Standard prediluted sample 8a 20 140 4 28 ug uL Assay Procedure corresponding to 5 uL H9 Holy Bradford original sample Bio Rad Protein Assay Microassa 8b a Not recommended Procedure Bradford Method tested in MN laboratory for compatibility with PSB PSB TCEP and Laemmli buffer protein samples Method compatible with PSB TCEP protein samples refering to manufacturer s product information Not tested in MN laboratories MACHEREY NAGEL 06 2014 Rev 05 33 Total DNA RNA and protein isolation Quantification of protein within the column flow through Alternative to quantification of protein dissolved in PSB TCEP protein can be quantified within the ethanolic lysate column flow through Knowlege of protein concentration in the column flow through helps to choose an appropriate volume for subsequent precipitation with Protein Precipatator PP The following methods are suitable to quantify protein in the column flow through 1 Bio Rad Protein Assay Bradford The standard assay procedure is compatibel with the column flow through however protein standards have to be prepared in a ethanolic lysate buffer mix Buffer RP1 and ethanol 70 in a ratio of 1 1 The microassay procedure is not compatible the column flow through 2 Pierce BCA Protein Assy Kit reducing agent compatible This met
49. shed catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG RNAlater is a registered trademark of AMBION Inc SPN is a registerede trademark of G Biosciences All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention pr
50. symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen MACHEREY NAGEL 06 2014 Rev 05 19 Total DNA RNA and protein isolation H 412 EUH031 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Precaution phrases P 210 P 233 P 260 P 261 P 273 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 330 P 332 313 P 333 313 P 337 313 P 342 311 P 403 235 P 363 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe vapours Dampf nicht einatmen Avoid breathing dust Einatmen von Staub vermeiden Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to f
51. t one side of the inner wall of the centrifuge tube e g for leaf and liver samples respectively If no precipitate is visible mark the side of the tube where a precipitate is expected in order to avoid touching this side of the inner tube wall with the pipette tip during the washing step See also section 2 4 how to avoid very large protein pellets 14 Dry protein pellet Dry precipitate for 5 10 min at room temperature keep lid open Note Large pellets e g complete precipitation of 700 uL column flow through from a 30 mg liver sample need longer time for drying Samples which are dried incomplete may cause problems when loading the sample onto the gel due to residual ethanol content No problems with over drying have been observed with small sized pellets See also section 2 4 how to avoid very large protein pellets 26 MACHEREY NAGEL 06 2014 Rev 05 NucleoSpin TriPrep 15 Prepare protein sample Add 20 100 pL PSB TCEP Protein Solving Buffer containing reducing agent Assure that PSB TCEP is clear not turbid If necessary warm PSB TCEP to gt 25 C to dissolve turbidity See section 2 4 as guideline for choosing an appropriate amount of PSB TCEP for dissolving of protein pellets Disaggregate large and visible pellets with a pipette tip to facilitate subsequent protein dissolution this is not necessary for small and invisible pellets Incubate for 3 min at 95 98 C for complete protein
52. table up to 6 months Frozen samples in Buffer RP1 should be thawed slowly before starting with the isolation of total RNA Wear gloves at all times during the preparation Change gloves frequently eril ly B IEMase are collected by centrifugation and directly lysed by adding Buffer RP1 according to step 2 of the standard protocol see section 5 1 Cell lysis of adherent growing cells in a culture dish Completely aspirate cell culture medium and continue immediately with the addition of Lysis Buffer RP1 to the cell culture dish Avoid incomplete removal of the cell culture medium in order to allow full lysis activity of the lysis buffer To trypsinize adherent growing cells Aspirate cell culture medium and add an equal amount of PBS in order to wash the cells Aspirate PBS Add 0 1 0 3 trypsin in PBS and incubate for an appropriate time to detach the cells from the dish surface After cell detachment add medium transfer cells to an appropriate tube not supplied and pellet by centrifugation for 5 min at 300 x g Remove supernatant and continue with the addition of lysis buffer to the cell pellet OLLI URAC ULL L ll EUREI 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 essential for efficient RNA preparation that all RNA contained in the sample is released from the cells by d
53. te protein Transfer an appropriate amount 10 700 uL of flow though into a fresh Collection Tube 1 5 mL supplied See section 2 4 as guideline for choosing an appropriate amount Add one volume PP Protein Precipitator Mix vigorously Incubate mixture at room temperature for approximately 10 min Note For samples of moderate to high protein content e g 100 mg young plant leaf 30 mg liver this incubation step may be omitted For samples of low to medium protein content e g 15mg young plant leaf the 10 min incubation increases protein yield relative to no incubation significantly An incubation of longer than one hour does not further increase protein yield Centrifuge for 5 min at 11 000 x g 13 Wash protein Remove supernatant by pipetting or decanting as complete as possible Add 500 uL of 50 ethanol to the pellet mixing or incubation at this step is not necessary Centrifuge 1 min at 11 000 x g Remove supernatant by pipetting or decanting as completely as possible Note Protein precipitate at this stage is quite different in appearance depending on kind and amount of starting material The consistence of precipitate might appear as no visible pellet e g for 10 000 cells 0 3 mg liver and 1 mg leaf samples a greenish tube wall coating on one side of the tube for e g leaf material green or white pellet at the bottom of the tube e g for leaf and liver samples respectively green or white crumbs a
54. tities of sample material depending on cell or tissue type in the range of 1 x 10 cells or 10 mg of tissue resulting in about 20 ug of RNA Protein characteristics Small 17 kDa to large 250 kDa proteins as well as glycoproteins membrane proteins lipoproteins phosphorylated proteins and structural proteins have been analyzed successfully The isolated protein is ready to use for SDS PAGE Western Blot analysis and protein quantification with the Protein Quantification Assay see ordering information MACHEREY NAGEL 06 2014 Rev 05 Total DNA RNA and protein isolation Typical yields of DNA RNA and protein DNA RNA protein yield ug 1000 100 Protein RNA DNA 0 1 10 000 100 000 HeLa cell number pieces 1 000 000 Figure 1 DNA RNA and protein yield from different amounts of HeLa cells DNA RNA and protein were isolated from different amounts of HeLa cells For cell numbers exceeding 250 000 only a fraction of the protein solution was used for precipitation and quantification total protein yield was calculated DNA RNA and protein were isolated as described in Figure 1 Obtained correlation coefficients between HeLa sample amount and DNA RNA and protein yield are shown in Table 2 Table 2 Correlation between sample amount nucleic acid and protein yield 3 x 104 5 x 10 cells 3x 104 1 x 10 cells DNA gt 0 99 gt 0 95 RNA
55. ty instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze RP1 Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 RA2 Guanidinium thiocyanate Warning 226 302 210 233 260 30 60 ethanol 20 412 273 301 312 35 EUH031 330 403 235 Guanidiniumthiocyanat Achtung 30 60 Ethanol 20 35 TCEP Tris 2 carboxyethyl Warning 315 319 280 302 352 phosphine hydrochloride lt gt 305 351 338 70 100 332 313 Tris 2carboxy ethyl phoshine Achtung 337 313 Hydrochlorid 70 100 rDNase rDNase lyophilized Danger 317 334 261 280 RNase free rDNase lyophilisiert Gefahr 302 352 304 340 333 313 342 311 363 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma
56. v 05 Total DNA RNA and protein isolation Quantification of protein dissolved in sample buffer Protein Quantification Assay MACHEREY NAGEL For most reliable results and convenience we recommend the MACHEREY NAGEL Protein Quantification Assay to quantify protein dissolved in PSB PSB TCEP or Laemmli buffer for ordering information see section 6 4 Alternatively the Karlsson protein quantification can be performed Protein quantification by Karlsson The procedure presented below based on the publication of Karlsson et al 1994 is also suitable for quantification of protein dissolved in Protein Solving Buffer PSB TCEP and may be followed alternatively Nucleic acids disturb protein quantification as described by Karlsson et al 1994 Protein samples obtained with the NucleoSpin RNA Protein kit are virtually free of nucleic acids thus protein quantification is not affected Upon addition of TCA Trichloracetic acid to the sample protein precipitates and causes turbidity The degree of turbidity is used for quantification relative to a sample with known protein concentration This test enables determination of protein concentration in the range 5 ng uL 20 ug uL by using variable sample volumes of 1 60 UL Recommended sample volume For protein Protein amount protein dissolved in PSB TCEP concentration in the per well range of 60 uL 0 01 0 33 ug uL 0 6 20 ug 20 uL 0 03 1 00 ug uL 0 6 20 ug 1 jL 0 6 20 ug uL 0 6 20 ug

DNA, RNA, and protein purification - MACHEREY

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