Genomic DNA and RNA purification User manual
Contents
1. MACHEREY NAGEL 03 2013 Rev 06 11 Genomic DNA and RNA purification NucleoBond NucleoBond AXG Columns AXG 20 AXG 100 AXG 500 are available separately as well and can be combined with a NucleoBond Buffer Set III to purify high molecular weight genomic DNA from bacteria and yeast or with NucleoBond Buffer Set IV to purify genomic DNA from tissue NucleoBond AXR and AXG Columns are polypropylene columns containing NucleoBond AX Silica Resin packed between two inert filter elements The columns are available in several sizes to accommodate a wide range of purification needs see Table 3 Table 3 NucleoBond Column binding capacities NucleoBond Columns Binding capacity AXR 80 80 ug RNA AXR 400 400 ug RNA AXG 20 20 ug genomic DNA AXG 100 100 ug genomic DNA AXG 500 500 ug genomic DNA All NucleoBond Columns are resistant to organic solvents such as alcohol chloroform and phenol and are free of DNase and RNase NucleoBond AX Resin can be used over a wide pH range from pH 2 5 8 5 and can remain in contact with buffers for up to three hours without any change in its chromatographic properties After three hours nucleic acids will begin to elute at increasingly lower salt concentrations Normally the resin remains functional in buffers containing up to 2 M salt It remains intact in the presence of denaturing agents like formamide urea or common detergents such as Triton X 100 and NP 40 3 3 Buffer2. IF INHALED If breathing is difficult remove to fresh air and keep at rest in a position comfortable for breathing Bei Einatmen Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhig stellen die das Atmen erleichtert If skin irritation occurs Get medical advice attention Bei Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doctor phy sician Bei Symptomen der Atemwege Giftinformationszentrum oder Arzt anrufen 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 18 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond RNA DNA 6 6 1 Protocols for NucleoBond RNA DNA 80 400 Isolation of RNA and genomic DNA from bacteria yeast and small amounts of eukaryotic cells Before starting the preparation Check if Buffer R4 was prepared according to section 4 Prepare lysozyme solution for RNA genomic DNA from bacteria 250 1000 ug mL TE pH 8 0 Prepare lyticase zymolase solution for RNA genomic DNA from yeast 1 M sorbitol 100 mM EDTA 14 mM B mercaptoethanol 50 100 U mL lyticase or zymolase pH 7 4 Check that isopropanol B mercaptoethanol and 85 ethanol is available AXR 80 AXR 400 Sample preparation Note During the homogenization process the isolated genomic D
3. Genomic DNA and RNA purification User manual NucleoBond RNA DNA NucleoBond CB NucleoBond AXG Columns March 2013 Rev 06 MACHEREY NAGEL MN www mn net com Genomic DNA and RNA purification NucleoBond Table of contents 1 Components 1 1 Kit contents 1 2 Reagents and equipment to be supplied by user Introduction 2 1 Properties 2 2 About this user manual Product description 3 1 The basic principle 3 2 Kit specifications 3 3 Buffer compositions Storage conditions and preparation of working solutions Safety instructions 5 1 Risk and safety phrases 5 2 GHS classification Protocols for NucleoBond RNA DNA 80 400 6 1 Isolation of RNA and genomic DNA from bacteria yeast and small amounts of eukaryotic cells 6 2 Isolation of RNA and genomic DNA from eukaryotic cells and tissue 6 3 RNA clean up of liquid samples and reaction mixtures Protocols for NucleoBond CB 20 100 500 Isolation of genomic DNA from blood and cell cultures Protocols for NucleoBond AXG Columns and NucleoBond Buffer Set III IV 8 1 Isolation of genomic DNA from bacteria 8 2 Isolation of genomic DNA from yeast 8 3 Isolation of genomic DNA from tissue Appendix 9 1 Troubleshooting 9 2 Ordering information 9 3 Product use restriction warranty 11 11 11 12 14 16 16 17 19 19 23 27 29 29 31 31 34 37 39 39 41 42 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purif
4. The volumes of the respective buffers used for a particular column size are highlighted Each procedural step is arranged like the following example taken from section 8 3 AXG 20 AXG 100 AXG 500 1 Cell dispruption Thoroughly homogenize the tissue mechanically Ultra Turrax in Buffer G2 Alternatively the tissue can be homogenized with a mortar and pestle under liquid nitrogen The fine powder is dissolved in Buffer G2 Note Homogenize the tissue as good as possible This step is very important for the lysis procedure as well as for a good flow rate of the NucleoBond AXG Columns For each step the name of the buffer buffer volume incubation times repeats or important handling steps are emphasized in bold type within the instruction Additional notes or optional steps are printed in italic In the example shown above the tissue sample is mechanically disrupted in 2 mL Mini prep with AXG 20 Columns 10 mL Midi prep with AXG 100 Columns or 20 mL Maxi prep with AXG 500 Columns of Buffer G2 MACHEREY NAGEL 03 2013 Rev 06 9 Genomic DNA and RNA purification NucleoBond Table 1 Protocol overview Sample type Sample size Purification of Section NucleoBond RNA DNA Bacterial cells 0 5 x 10 AXR 80 RNA and DNA 6 1 2x 10 AXR 400 Yeast 10 AXR 80 RNA and DNA 6 1 109 1019 AXR 400 Eukaryotic cells 10 10 AXR 80 RNA and DNA 6 1 108
5. Isolation of genomic DNA from yeast For the isolation of genomic DNA from yeast MACHEREY NAGEL does not offer ready to use kits The columns and the most important buffers can be ordered separately Our NucleoBond Buffer Set Ill REF 740603 contains all necessary buffer solutions Buffers G3 G4 N2 N3 and N5 Proteinase K and RNase A Sorbitol buffer as well as lyticase or zymolase stock solution have to be prepared fresh These two enzymes are not included in the buffer set Before starting the preparation Check if Buffer R4 was prepared according to section 4 Prepare lyticase zymolase solution for total DNA from yeast 1M sorbitol 100 mM EDTA 14 mM B mercaptoethanol 50 100 U mL lyticase or zymolase pH 7 4 Check that isopropanol and 70 96 ethanol is available AXG 20 AXG 100 AXG 500 1 Cell disruption Resuspend the yeast cell pellet in lyticase zymolase solution and incubate at 30 C for 30 min 600 uL lyticase zymolase 3 mL 15 mL lyticase lyticase zymolase zymolase Centrifuge the mixture for 10 min at 5 000 x gto pellet the spheroblasts Remove the supernatant and resuspend the cell pellet in Buffer G3 by vortexing 1mL 4 mL 12 mL Add Proteinase K stock solution Incubate the mixture at 37 C 25 uL 100 uL 450 pL 20 min 40 min Add Buffer G4 and mix by vortexing 400 uL 1 2mL 4 mL 34 MACHEREY NAGEL 03 2013 R
6. 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 published 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 co
7. compositions Table 4 NucleoBond Buffer compositions Buffer Buffering compounds G1 320 mM saccharose 5 mM MgCl 10 mM Tris HCl 196 Triton X 100 pH 7 5 G2 800 mM GuHCI 30 mM EDTA 30 mM Tris HCI 5 Tween 20 0 5 Triton X 100 pH 8 0 G3 50 mM EDTA 50 mM Tris HCl 0 5 Tween 20 0 5 96 Triton X 100 pH 8 0 G4 3 M GuHCl 20 Tween 20 pH 5 5 12 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond Table 4 NucleoBond Buffer compositions Wa 50 mM Tris acetate 4 M GuSCN pH 7 8 W3 20 Triton X 100 W4 3 M sodium acetate pH 6 5 W5 10 mM Tris acetate 1 mM EDTA pH 7 8 W6 200 mM Tris acetate 1 5 M KCI pH 6 3 RO 100 mM Tris acetate 15 96 ethanol pH 6 3 R1 100 mM Tris acetate 15 96 ethanol 400 mM KCI pH 6 3 R2 100 mM Tris acetate 15 96 ethanol 900 mM KCI pH 6 3 R3 100 mM Tris acetate 15 ethanol 1150 mM KCI pH 6 3 R4 100 mM Tris acetate 15 96 ethanol 1150 mM KCI 6 M urea pH 6 3 N2 100 mM Tris H4PO 1596 ethanol 900 mM KCI pH 6 3 0 15 Triton X 100 N3 100 mM Tris H4PO 15 ethanol 1150 mM KCI pH 6 3 N5 100 mM Tris H4PO 15 ethanol 1000 mM KCI pH 8 5 Note Keep all buffer bottles tightly closed The concentration of KCI required for eluting the desired nucleic acid is highly dependent on the pH value of the eluent Figure 2 For this reason pH values must be carefully controlled if the buffers have been prepared by
8. 5 x 109 AXR 400 105 5 x 10 AXR 80 RNA and DNA 6 2 5 x 109 2 x 10 AXR 400 Tissue 20 mg AXR 80 RNA and DNA 6 2 100 mg AXR 400 Liquid samples 100 uL AXR 80 RNA and DNA 6 3 reaction mixtures 400 uL AXR 400 NucleoBond CB Eukaryotic cells 5 x 109 AXG 20 DNA 7 1 2x 107 AXG 100 108 AXG 500 Whole blood 0 1 1 mL AXG 20 DNA 74 2 5 mL AXG 100 5 20 mL AXG 500 Buffy coat 50 uL AXG 20 DNA 7 1 250 uL AXG 100 1 mL AXG 500 NucleoBond AXG NucleoBond Buffer Set Ill Bacteria 2 4 mL AXG 20 DNA 8 1 15 20 mL AXG 100 60 80 mL AXG 500 Yeast 10 AXG 20 DNA 8 2 109 1019 AXG 100 1019 101 AXG 500 NucleoBond AXG NucleoBond Buffer Set IV Tissue 20 mg AXG 20 DNA 8 3 100 mg AXG 100 400 mg AXG 500 10 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 3 Product description 3 4 The basic principle NucleoBond RNA DNA and NucleoBond CB as well as NucleoBond AXG in combination with a NucleoBond Buffer Set employ chaotropic salt or enzymatic lysis procedures to prepare a variety of sample materials for genomic DNA and RNA purification After equilibrating the appropriate NucleoBond Column RNA and or DNA are bound to the anion exchange resin under low salt conditions at an acidic pH of 6 3 Whereas RNAis digested for the purification of genomic DNA with NucleoBond AXG and NucleoBond Buffer Set several different RNA species can be specifically washed o
9. NA can be sheared into small fragments and may partially appear in the RNA fraction If a complete removal of genomic DNA is required a DNase treatment after the precipitation of RNA or for example a subsequent LiCI precipitation of the isolated nucleic acids is recommended For the isolation of RNA from bacteria An enzymatic treatment before starting the isolation is recommended For this purpose add for example Iysozyme to the bacterial cell pellet resuspend it and incubate for 10 min at room temperature Depending on the bacterial strain other appropriate enzymes are also compatible with this method 100 uL 400 uL Afterwards add Buffer W1 and mix carefully For the isolation of RNA from yeast Resuspend the yeast cell pellet in lyticase zymolase solution and incubate for 30 min at 30 C 1 mL 4 mL lyticase lyticase ymolase zymolase MACHEREY NAGEL 03 2013 Rev 06 19 NucleoBond RNA DNA AXR 80 AXR 400 Centrifuge for 10 min at 1 000 x g to pellet the spheroblasts Remove the supernatant add Buffer W1 to the cell pellet and homogenize the lysis mixture by vortexing 500 uL 2 mL For the isolation of RNA from eukaryotic cells Add Buffer W1 to the cells Homogenize the lysis mixture by pipetting up and down vortexing by using a machanical disruption device e g a PTFE homogenizer Cell lysis and separation of proteins Add B mercap
10. NucleoBond 1 1 Kit contents continued NucleoBond NucleoBond Buffer Set Ill Buffer Set IV Application Genomic DNA Genomic DNA from bacteria and yeast from tissue REF 740603 740604 Buffer G2 2 x 125 mL Buffer G3 150 mL S Buffer G4 40 mL Buffer N2 2x 125 mL 2 x 125 mL Buffer N3 250 mL 250 mL Buffer N5 120 mL 120 mL RNase A lyophilized 30 mg 2x 25mg Proteinase K lyophilized 2x 50 mg 2x 50 mg Proteinase Buffer PB 8 mL 8 mL User manual 1 1 For preparation of working solutions and storage conditions see section 4 6 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 1 1 Kit contents continued NucleoBond NucleoBond NucleoBond AXG 20 AXG 100 AXG 500 REF 740544 740545 740546 NucleoBond AXG 20 20 Columns NucleoBond AXG 100 20 Columns NucleoBond AXG 500 10 Columns Plastic Washer 10 10 5 User manual 1 1 1 1 2 Reagents and equipment to be supplied by user Reagents B mercaptoethanol Isopropanol room temperatured 85 or 7096 ethanol room temperatured depending on protocol Buffer for reconstitution of DNA for example TE buffer or sterile H O Lysozyme for the isolation of RNA DNA from bacteria Lyticase zymolase for the isolation of RNA DNA from yeast Please see the introduction of the related protocol for more detailed information Equipment Refrigerated centrifuge capable of reaching gt 5 000 x g with rotor for t
11. 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 N VITRO diagnostic use Please pay attention to the package of the product N VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR 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
12. an organic solvent of low viscosity Increase buffer volume Strong white pellet after precipitation Coprecipitation of the salt Check the purity of the isopropanol Perform precipitation at room centrifugation temperature except Do not let the eluate drop directly into a vial with isopropanol 40 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 9 2 Ordering information Product REF Pack of NucleoBond RNA DNA 80 740650 25 preps NucleoBond RNA DNA 400 740651 10 preps NucleoBond CB 20 740507 20 preps NucleoBond CB 100 740508 20 preps NucleoBond CB 500 740509 10 preps NucleoBond Buffer Set III 740603 1 set NucleoBond Buffer Set IV 740604 1 set NucleoBond AXG 20 740544 20 columns NucleoBond AXG 100 740545 20 columns NucleoBond AXG 500 740546 10 columns NucleoBond Xtra Combi Rack 740415 1 NucleoBond Rack Large 740563 1 rDNase Set 740963 1 set RNase A 740505 50 50 mg 740505 100 mg MACHEREY NAGEL 03 2013 Rev 06 41 Genomic DNA and RNA purification NucleoBond 9 3 Product use restriction warranty NucleoSpin RNA DNA CB AXG 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
13. as a good flow rate of the column an overloading of the column must be avoided For the first time it is better to start using a low cell number AXG 20 4 x 10 AXG 100 2 x 10 AXG 500 1 x 10 cells can be increased stepwise If bacteria are used that contain plasmid DNA and genomic DNA start with half of the culture volume recommended for non plasmid containing bacteria Before starting the preparation Check if Buffer G3 and Proteinase K were prepared according to section 4 If lysozyme is required redissolve it in sterile or ddH O 100 mg mL The solution should be divided in aliquots and stored at 20 C AXG 20 AXG 100 AXG 500 1 Cell disruption Pellet the bacterial cells from an appropriate volume of culture by centrifugation at 3 000 5 000 x g for 10 min Discard the supernatant Resuspend the bacterial pellet in Buffer G3 by vortexing MACHEREY NAGEL 03 2013 Rev 06 31 NucleoBond AXG and NucleoBond Buffer Set AXG 20 AXG 100 AXG 500 Add the ysozyme optional and the Proteinase K stock solution 20 uL 80 uL 300 uL lysozyme lysozyme lysozyme 25 HL 100 uL 450 uL Proteinase K Proteinase K Proteinase K Incubate the mixture at 37 C 20 min 40 min ETIN Add Buffer G4 and mix by vortexing 400 pL 1 2 mL 4 mL Incubate the mixture at 50 C for 30 min If the lysate is not clear after incubation
14. ates and other unwanted cellular components The purified nucleic acid products are suitable for use in the most demanding molecular biology applications including transfection in vitro transcription automated or manual sequencing cloning hybridization and PCR Plasmid DNA large constructs Single stranded DNA Double stranded DNA mRNA 168 238 rRNA 5S rRNA Compound class tRNA emn Proteins dyes polysaccharides metabolites trinucleotides rRNA large constructs Plasmid DNA Absorbance at 260 nm 0 0 5 1 1 5 Salt concentration for elution M KCI Figure 1 Elution profile of NucleoBond AX Resin at pH 7 0 The more interactions a nucleic acid can form between the phosphate backbone and the positively charged resin the later it is eluted with increasing salt concentration Large nucleic acids carry more charges than short ones double stranded DNA more than single stranded RNA 8 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 2 2 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoBond RNA DNA or NucleoBond CB kits or NucleoBond AXG in combination with a NucleoBond Buffer Set is used for the first time All technical literature is available on the internet at www mn net com The protocols in this manual see overview in Table 1 are organized as follows
15. ch bottle to each bottle 20 mg 40 mg 40 mg Add 1 mL Add 2 mL Add 2 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer PB PB NucleoBond Buffer Set III NucleoBond Buffer Set IV REF 740603 740604 Buffer G2 _ 2x125 mL Add 25 mg RNase A to each bottle Buffer G3 150 mL _ Add 30 mg RNase A Proteinase K 2x50 mg 2x50 mg Add 2 5 mL Proteinase Buffer PB to each vial Add 2 5 mL Proteinase Buffer PB to each vial MACHEREY NAGEL 03 2013 Rev 06 15 Genomic DNA and RNA purification NucleoBond 5 Safety instructions The following components of the NucleoBond kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 5 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol G4 Guanidine hydrochloride x Xn R 22 Guanidinhydrochlorid 36 38 Proteinase K Proteinase K lyophilized x Xn R 36 37 38 S 22 24 Proteinase K lyophilisiert 42 26 36 37 RNase A RNase A lyophilized x Xn R 42 43 S 22 24 RNase A lyophilisiert Wa Guanidinium thiocyanate x Xn R 32 Guanidiniumthiocyanat Risk phrases R22 R32 R 36 37 38 R 36 38 R42 R 42 43 Harmful if swallowed Gesundheitssch dlich beim Verschlucken Contact with acids liberates very toxic gas Entwickelt bei Ber hrung mit S ure sehr giftige Gase Irritating to eyes res
16. ev 06 NucleoBond AXG and NucleoBond Buffer Set AXG 20 AXG 100 AXG 500 Incubate the mixture at 50 C for 30 min If the lysate is not clear after incubation with Proteinase K the incubation time should be prolonged If any insoluble cell components are observed the sample should be clarified by a short centrifugation 5 000 x g 5 min Note It is very important to obtain a clear lysate in order to avoid clogging of the column Equilibration Equilibrate the column with Buffer N2 1mL 2mL 5mL Binding Add Buffer N2 room temperature to the sample Vortex the mixture for 15 s at maximum speed Load the sample onto the column Allow it to enter the resin by gravity flow 1mL 5 mL 10 mL Wash Wash the column with Buffer N3 3x1mL 3x4mL 3x8mL Elution Elute the genomic DNA with Buffer N5 A second elution step with the same volume will increase the yield slightly 10 15 1mL 5 mL 8 mL Precipitation Add 0 7 volume of isopropanol room temperature mix incubate 30 60 min at room temperature and centrifuge at 4 C 15 000 rpm for 25 min 700 uL 3 5 mL 5 6 mL MACHEREY NAGEL 03 2013 Rev 06 35 NucleoBond AXG and NucleoBond Buffer Set AXG 20 AXG 100 AXG 500 If the pellet looks glassy air dry it not longer than 15 min and redissolve it in slightly alkaline buffer e g TE pH 8 overnight on a s
17. haker or at 55 C for 1 2 hours If a white pellet is obtained additionally wash it with 70 ethanol and redissolve it as described above 36 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond AXG and NucleoBond Buffer Set 8 3 Isolation of genomic DNA from tissue For the isolation of genomic DNA from tissue MACHEREY NAGEL does not offer ready to use NucleoBond kits The columns as well as the buffer solutions can be ordered separately Our NucleoBond Buffer Set IV REF 740604 contains all necessary buffer solutions Buffers G2 N2 N3 and N5 Proteinase K and RNase A Please note In order to achieve a high yield of DNA the tissue samples should be kept in liquid nitrogen at all time before the preparation If the tissue sample is treated with 20 6 glycerol or 20 DMSO centrifuge the sample and discard the supernatant Before starting the preparation Check if Buffer G2 and Proteinase K were prepared according to section 4 AXG 20 AXG 100 AXG 500 1 Cell disruption Thoroughly homogenize the tissue mechanically Ultra Turrax in Buffer G2 Alternatively the tissue can be homogenized with a mortar and pestle under liquid nitrogen The fine powder is dissolved in Buffer G2 Note Homogenize the tissue as good as possible This step is very important for the lysis procedure as well as for a good flow rate of the NucleoBond AXG Columns Transfer the homogenate to a 15 or 50 mL screw cap t
18. he ap propriate centrifuge tubes or bottles Centrifugation tubes or vessels with suitable capacity for the volumes specified in the respective protocol NucleoBond Rack Large NucleoBond Xtra Combi Rack see ordering infor mation or equivalent holder MACHEREY NAGEL 03 2013 Rev 06 7 Genomic DNA and RNA purification NucleoBond 2 Introduction 2 1 Properties NucleoBond AX is a patented silica based anion exchange resin developed by MACHEREY NAGEL for routine separation of different classes of nucleic acids NucleoBond AX Resin forms the basis for the entire line of nucleic acid purification products presented in this user manual NucleoBond AX Resin consists of hydrophilic macro porous silica beads coupled to a methyl ethylamine functional group The functional group provides a high overall charge density that permits the negatively charged phosphate backbone of RNA or DNA to bind with high specificity to the resin Due to a specialized manufacturing process that is rigorously controlled and monitored the beads are uniform in diameter and contain particularly large pores These special properties allow for optimum flow rates through the column and more efficient binding of nucleic acids to the matrix Thus using the matrix you can achieve sharp well defined elution profiles for individual nucleic acid species see Figure 1 NucleoBond AX can separate distinct nucleic acids from each other and from proteins carbohydr
19. ication NucleoBond 1 Components 1 1 Kit contents NucleoBond NucleoBond RNA DNA 80 RNA DNA 400 25 preps 10 preps REF 740650 740651 Buffer W1 20 mL 35 mL Buffer W3 10 mL 10 mL Buffer W4 15 mL 25mL Buffer W5 35 mL 80 mL Buffer W6 15 mL 25mL Buffer RO 2x125mL 4x 100 mL Buffer R1 2x120mL 2x100 mL Buffer R2 2x120mL 2x80mL Buffer R3 2x100mL 125 mL Buffer R4 75 mL 75mL Buffer N5 120 mL 120 mL Urea 36 25 g 36 25 g NucleoBond AXR 80 25 Columns NucleoBond AXR 400 10 Columns Plastic Washer 10 5 User manual 1 1 For preparation of working solutions and storage conditions see section 4 4 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 1 1 Kit contents continued NucleoBond NucleoBond NucleoBond CB 20 CB 100 CB 500 20 preps 20 preps 10 preps REF 740507 740508 740509 Buffer G1 27 mL 2x63 mL 2x 107 mL Buffer G2 25 mL 125 mL 125 mL Buffer N2 2x25 mL 150 mL 200 mL Buffer N3 3 x 30 mL 250 mL 250 mL Buffer N5 32 mL 120 mL 120 mL Saccharose 3 29 g 2x 7 70 g 2x 13 14g Proteinase K 20 mg 40 mg 40 mg lyophilized Proteinase Buffer PB 3 6 mL 3 6 mL 3 6 mL NucleoBond AXG 20 20 Columns NucleoBond AXG 100 20 Columns NucleoBond AXG 500 10 Columns Plastic Washer 10 10 5 User manual 1 1 1 For preparation of working solutions and storage conditions see section 4 MACHEREY NAGEL 03 2013 Rev 06 5 Genomic DNA and RNA purification
20. ifuge at 4 C 15 000 rpm for 25 min 700 uL 3 5 mL 5 6 mL If the pellet looks glassy air dry it not longer than 15 min and redissolve it in slightly alkaline buffer e g TE pH 8 overnight on a shaker or at 55 C for 1 2 hours If a white pellet is obtained additionally wash it with 70 ethanol and redissolve it as described above 30 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond AXG and NucleoBond Buffer Set 8 Protocols for NucleoBond AXG Columns and NucleoBond Buffer Set III IV 8 1 Isolation of genomic DNA from bacteria For the isolation of genomic DNA from bacteria MACHEREY NAGEL does not offer ready to use kits The columns as well as the buffer solutions can be ordered separately The NucleoBond Buffer Set Ill REF 740603 contains all necessary buffers Buffers G3 G4 N2 N3 and N5 Proteinase K as well as RNase A Lysozyme is not included in this buffer set Please note Gram positive bacteria are more difficult to lyse Reagents like lysozyme lysostaphin etc are recommended and compatible with this method When using clinical samples tissue or other innomogenous material for DNA isolation additional homogenisation techniques Ultra Turrax Dounce homogenisator etc in combination with an enzymatic digest lyticase lysozyme lysostaphin may be necessary In general follow our standard protocol for the isolation of genomic DNA from bacteria In order to obtain pure DNA as well
21. ion buffer or its pH are too low Adjust pH or prepare a new buffer MACHEREY NAGEL 03 2013 Rev 06 39 Genomic DNA and RNA purification NucleoBond Problem Possible cause and suggestions Column overloaded Do not overload the column because this will result in decreased yield and purity of RNA preparations If you are No clear RNA in doubt use first a small amount of sample in order to find separation out the RNA content Afterwards use the appropriate amount of sample according to the limited lysing capacity of the lysis buffer and according to the capacity of the column as indicated in the protocol Viscosity of the sample is too high Column Use larger buffer volumes for sample preparation Use a blocked prolonged centrifugation step to get a clear supernatant Mix the sample with one volume of equilibration buffer RNA RNase A digestion was insufficient contamination in DNA fraction Add more RNase Increase volume of wash buffer No nucleic acid after precipitation Nucleic acid pellet was lost Handle with care Nucleic acid was not resuspended Handle with care Nucleic acid was not precipitated Check organic solvent Mix the suspension and use a longer centrifugation time Insufficient resuspension of purified nucleic acid Nucleic acid was overdried Dissolve for a longer time at somewhat higher temperature Residual salt or organic solvent in the pellet Wash the pellet with
22. ion of proteins Sample preparation Note During the homogenization process the isolated genomic DNA can be sheared into small fragments and may partially appear in the RNA fraction If a complete removal of genomic DNA is required a DNase treatment after the precipitation of RNA or for example a subsequent LiCI precipitation of the isolated nucleic acids is recommended Add Buffer W1 to the cells or tissue Add B mercaptoethanol to the solution and homogenize 3 4 times for each 20 s using a commercial homogenizer e g Polytron Dounce Alternatively other homogenization tools like mortar and pestle in the presence of liquid nitrogen may be used In order to reduce the viscosity pass the lysate 3 times through a sterile plastic syringe fitted with a 20 gauge needle Add Buffer W3 Mix the sample and incubate for 15 min at 4 C 50 uL 200 pL Add Buffer W4 Mix the sample carefully and incubate it for 15 min at 4 C Centrifuge the mixture at 10 000 x g for 20 min at 4 C in order to separate cellular debris MACHEREY NAGEL 03 2013 Rev 06 23 NucleoBond RNA DNA AXR 80 AXR 400 2 Precipitation of nucleic acids Add isopropanol to the supernatant mix carefully and incubate for 10 min on ice Centrifuge the mixture at 10 000 x g for 20 min at 4 C Discard the supernatant and dissolve the RNA pellet i
23. itation Add isopropanol to the eluate mix incubate on ice for 15 min and centrifuge for 25 min at 10 000 x g and 4 C 5mL The RNA pellet is washed with 85 ethanol dried for 5 10 min and dissolved in an appropriate buffer for further use 1mL 1mL If complete removal of DNA is necessary for subsequent reactions an additional enzymatic treatment with DNase is recommended see ordering information for rDNase Set 28 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond CB 7 Protocols for NucleoBond CB 20 100 500 Isolation of genomic DNA from blood and cell cultures Before starting the preparation Check if Buffer G1 G2 and Proteinase K were prepared according to section 4 Before starting the procedure chill 20 mL ddH O on ice AXG 20 AXG 100 AXG 500 1 Cell disruption Cell culture After washing the cells twice with PBS and centrifugation resuspend the cells in PBS to a final concentration of 10 cells mL Add 1 volume of Buffer G1 ice cold and 3 volumes ddH O ice cold to 1 volume whole blood or cell suspension Example For 1 mL cell suspension 107 cells or 1 mL blood add 1 mL of Buffer G1 and 3 mL ddH O 1 vol G1 1 vol G1 1 vol G1 3 vol ddH O 3 vol ddH O 3 vol ddH O Mix the suspension by inverting the tube 6 8 times and incubate the mixture for 10 min on ice Centrifuge the mixture at 4 C important for 15 min at 1 300 1 500 x g arou
24. lt Gefahrstoff GHS Symbol H S tze P S tze G4 Guanidine hydrochloride Warning 24 36 Guanidinhydrochlorid 24 36 Achtung N2 N3 N5 Ethanol 5 20 Warning Ethanol 5 20 Achtung Proteinase K Proteinase K lyophilized Danger 334 261 Proteinase K lyophilisiert Gefahr 3044341 342 311 RO R1 R2 Ethanol 5 20 96 Warning R3 R4 Ethanol 5 20 96 D Achtung RNase A RNase A lyophilized Danger 317 334 261 304 341 RNase A lyophilisiert Gefahr 342 311 301 312 280 302 352 333 313 wi Guanidinium thiocyanate Warning 30 60 Guanidiniumthiocyanat Achtung 30 60 MACHEREY NAGEL 03 2013 Rev 06 17 Genomic DNA and RNA purification NucleoBond Hazard phrases H 317 H 334 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen Precaution phrases P 261 P 280 P 3014312 P 302 352 P 3044341 P 333 313 P 3424311 Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell Bei Verschlucken Bei Unwohlsein Giftinformationszentrum oder Arzt anrufen IF ON SKIN Wash with plenty of soap and water Bei Kontakt mit der Haut Mit viel Wasser und Seife waschen
25. n Buffer W1 very carefully 200 uL 800 uL Note If dissolution in Buffer W1 is not possible try one of the following options a Incubate at 65 C for 1 3 min Note that RNA might be damaged b Dissolve pellet in Buffer W5 and Buffer W6 according to section 6 3 step 1B Optional If total removal of DNA is necessary for subsequent reactions an additional enzymatic treatment with DNase is recommended see ordering information for rDNase Set Dissolve the pellet in Reaction Buffer for rDNase and follow the instructions given in the rDNase Set leaflet Finally add Buffer W1 and proceed as described above 50 uL 100 200 uL Optional Remove insoluble particles by centrifugation 10 min 12 000 x g 4 C and collect the supernatant Add Buffer RO to supernatant and mix 2mL 8 mL 3 Equilibration Equilibrate a NucleoBond AXR Column with Buffer R1 1mL 3 mL 24 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond RNA DNA AXR 80 AXR 400 Binding Transfer the clear supernatant of step 2 to the column Collect the flow through containing genomic DNA Wash Wash the NucleoBond AXR Column with Buffer R1 for purification of tRNA or tRNA mRNA rRNA or Buffer R1 2 1 1 for purification of rRNA mRNA or Buffer R2 for purification of rRNA or viral RNA stringent wash 6 mL 12 mL Elution Elute the RNA with Buffer R4 preheating to 50 C improves the yield of RNA but may increase DNA contaminati
26. nd 3 500 rpm Discard the supernatant A small red pellet is visible Add Buffer G1 ice cold and ddH O ice cold and resuspend the pellet by vortexing 5 10 s Centrifuge the mixture at 4 C important for 15 min at 1 300 1 500 x g around 3 500 rpm Discard the supernatant The pellet should be almost white 200 pL G1 1mL G1 2mL G1 750 uL ddH O 3 mL ddH O 6 mL ddH O Small red spots on the pellet are not critical for the procedure If the whole pellet is slightly red repeat this washing step Add Buffer G2 and completely resuspend the pellet by vortexing for 15 30 s MACHEREY NAGEL 03 2013 Rev 06 29 NucleoBond CB AXG 20 AXG 100 AXG 500 Add Proteinase K 20 mg mL and incubate the mixture for 60 min at 50 C 50 uL 100 pL 200 uL 2 Equilibration Equilibrate the NucloBond AXG Column with Buffer N2 1mL 2mL 5 mL 3 Binding Add Buffer N2 room temperature to the sample Vortex the mixture for 15 s at maximum speed Load the sample onto the column Allow it to enter the resin by gravity flow 4 Wash Wash the column with Buffer N3 3x1mL 3x4mL 5 Elution Elute the genomic DNA with Buffer N5 A second elution step with the same volume of elution buffer will increase the yield slightly 15 20 96 1mL 5mL 8 mL 6 Precipitation Add 0 7 volume of isopropanol room temperature mix incubate 30 60 min at room temperature and centr
27. ntact 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 2 mn net com Trademarks disclaimer NucleoBond is a trademark of MACHEREY NAGEL GmbH amp Co KG 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 products 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 MACHEREY NAGEL 03 2013 Rev 06 43
28. olution is stable at 20 C for at least 6 months Before starting the first NucleoBond Buffer Set III IV purification prepare the following Buffer G2 NucleoBond Buffer Set IV Add 1 mL Buffer G2 to an RNase A vial and vortex Transfer the resulting solution back into the Buffer G2 bottle and mix thoroughly Indicate date of RNase A addition Store Buffer G2 containing RNase A at 4 C The solution will be stable at this temperature for at least 6 months Buffer G3 NucleoBond Buffer Set Ill Add 1 mL Buffer G3 to an RNase A vial and vortex Transfer the resulting solution back into the Buffer G3 bottle and mix thoroughly Indicate date of RNase A addition Store Buffer G3 containing RNase A at 4 C The solution will be stable at this temperature for at least 6 months Proteinase K Add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for at least 6 months 14 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond NucleoBond RNA DNA 80 NucleoBond RNA DNA 400 25 preps 10 preps REF 740650 740651 Buffer R4 75 mL 75 mL Add 36 25 g Urea Add 36 25 g Urea REF Buffer G1 Proteinase K NucleoBond CB NucleoBond CB NucleoBond CB 20 500 20 preps 20 preps 10 preps 740507 740508 740509 27 mL 2x63 mL 2 x 107 mL Add 3 29 g Add 7 70 g Add 13 14 g Saccharose Saccharose Saccharose to ea
29. on Alternatively use Buffer R3 for elution to reduce DNA contamination Yield however can be reduced as well 3 mL 6 mL Optional subsequent isolation of genomic DNA Apply the flow through step 4 to the column and wash the column with Buffer R3 to remove residual RNA i 3mL mL Elute DNA with Buffer N5 preheated to 50 C 3mL 6 mL Precipitation Add isopropanol to the RNA DNA eluate mix incubate for 15 min on ice and centrifuge for 25 min at 10 000 x g and 4 C 2 5 mL 5mL MACHEREY NAGEL 03 2013 Rev 06 25 NucleoBond RNA DNA AXR 80 AXR 400 Wash the RNA DNA pellet with 85 ethanol dry the pellet for 5 10 min and dissolve it in an appropriate buffer for further use 1mL 1 mL If complete removal of DNA or RNA is necessary for subsequent reactions an additional enzymatic treatment with DNase or RNase is recommended see ordering information for rDNase Set or RNase Alternatively a further purification with NucleoBond or NucleoSpin kits is recommended 26 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond RNA DNA 6 3 RNAclean up of liquid samples and reaction mixtures AXR 80 AXR 400 1 Sample preparation A For RNA containing fluid samples or reaction mixtures Use the indicated sample volumes If doubled volumes have to be processed use doubled volumes of Buffers W1 W3 and RO 10 100
30. ontamination Alternatively use Buffer R3 for elution to reduce DNA contamination Yield however can be reduced as well 3 mL 6 mL Optional subsequent isolation of genomic DNA Apply the flow through step 4 to the column and wash the column with Buffer R3 to remove residual RNA 3 mL 6 mL Elute DNA with Buffer N5 preheated to 50 C 3 mL 6 mL MACHEREY NAGEL 03 2013 Rev 06 21 NucleoBond RNA DNA AXR 80 AXR 400 Precipitation Add isopropanol to the RNA DNA eluate mix incubate for 15 min on ice and centrifuge for 25 min at 10 000 x g and 4 C 2 5 mL 5 mL 25mL Wash the RNA DNA pellet with 85 ethanol dry the pellet for 5 10 min and dissolve it in an appropriate buffer for further use 1mL 1mL If complete removal of DNA or RNA is necessary for subsequent reactions an additional enzymatic treatment with DNase or RNase is recommended see ordering information for rDNase Set or RNase Alternatively a further purification with NucleoBond or NucleoSpin kits is recommended 22 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond RNA DNA 6 2 Isolation of RNA and genomic DNA from eukaryotic cells and tissue Before starting the preparation Check if Buffer R4 was prepared according to section 4 Check that isopropanol B mercaptoethanol and 85 ethanol is available AXR 80 AXR 400 Cell lysis and separat
31. piratory system and skin Reizt die Augen Atmungsorgane und die Haut Irritating to eyes and skin Reizt die Augen und die Haut May cause sensitization by inhalation Sensibilisierung durch Einatmen m glich May cause sensitization by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich Hazard labeling not necessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further infor mation see Material Safety Data Sheet 16 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond Safety phrases 22 Do not breathe dust Staub nicht einatmen S24 Avoid contact with the skin Ber hrung mit der Haut vermeiden S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren S 36 37 Wear suitable protective clothing and gloves Bei der Arbeit geeignete Schutzhandschuhe und Schutzkleidung tragen 5 2 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 Inha
32. t on a shaker or at 55 C for 1 2 hours If a white pellet is obtained additionally wash it with 70 ethanol and redissolve it as described above 38 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 9 Appendix 9 1 Troubleshooting If any problems with the preparation arise proceed as follows In order to get an idea of what has been the problem please collect the flow through the wash and the eluate fraction Precipitate the fractions and load them on an agarose gel In combination with this troubleshooting guide this will help to solve your problem Problem Possible cause and suggestions Salt concentration of the sample is too high Dilute the sample or precipitate and redissolve it pH value of the sample is higher than pH 6 5 Adjust the pH of the sample pH or salt concentrations of buffers are too high e Adjust pH or prepare new buffers No quantitative adsorption of nucleic acids High viscosity sample Increase the volume of sample preparation buffers to reduce viscosity Column was overloaded with nucleic acid Use a bigger column or purify excess DNA on a new column No nucleic acid in the sample Check the pH of all buffers used and repeat the purification No nucleic acid adsorbed See above No elution of Salt concentration or pH of the washing buffer are too high RNA dsDNA or ssDNA Adjust pH or prepare a new buffer Salt concentration of the elut
33. the customer A deviation of more than 0 1 pH unit from the given values may affect yields If you are consistently experiencing reduced yields check the pH of all buffers before continuing Buffers should be adjusted with acidic acid H3PO or KOH T T 1 5 41 5 KCI M 1 25 11 25 1 0 41 0 0 75 0 75 0 5 40 5 I li l l li 6 0 6 5 7 0 7 5 pH Figure 2 Elution profile of nucleic acids depending on the pH and salt concentration of the elution buffer 100 mM Tris H PO 15 ethanol MACHEREY NAGEL 03 2013 Rev 06 13 Genomic DNA and RNA purification NucleoBond 4 Storage conditions and preparation of working solutions All kit components can be stored at room temperature 18 25 C and are stable for at least one year Before starting the first NucleoBond RNA DNA purification prepare the following Buffer R4 Add 75 mL of Buffer R4 to 36 25 g Urea and mix thoroughly Transfer all of the resulting Buffer R4 with Urea back to the Buffer R4 bottle Indicate date of Urea addition The solution will be stable at this temperature for at least 6 months Before starting the first NucleoBond CB purification prepare the following Buffer G1 Add Saccharose to Buffer G1 After addition of Saccharose to Buffer G1 the buffer has to be stored at 4 C and is stable for at least 3 months Proteinase K Add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K s
34. 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 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 42 MACHEREY NAGEL 03 2013 Rev 06 Genomic DNA and RNA purification NucleoBond 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
35. toethanol to the solution and homogenize by vortexing In order to reduce the viscosity pass the lysate 3 times through a sterile plastic syringe fitted with a 20 gauge needle 0 5 uL 2 uL Add Buffer W3 mix the sample and incubate for 5 min at 4 C 50 uL 200 pL Add Buffer W4 mix the sample carefully and incubate for 5 min at room temperature Centrifuge the mixture at 10 000 x g for 20 min at 4 C in order to separate cellular debris and proteins 500 uL mL Add Buffer RO to the supernatant and mix carefully 10 mL 36 mL Optional If necessary centrifuge the solution 10 min 12 000 x g 4 C in order to remove insoluble particles and to avoid clogging of the column and collect the clear supernatant 20 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond RNA DNA AXR 80 AXR 400 Equilibration Equilibrate a NucleoBond AXR Column with Buffer R1 1mL 3 mL Binding Transfer the clear supernatant of step 2 to the column Collect the flow through containing genomic DNA Wash Wash the NucleoBond AXR Column with Buffer R1 for purification of tRNA or tRNA mRNA rRNA or Buffer R1 2 1 1 for purification of rRNA mRNA or Buffer R2 for purification of rRNA or viral RNA stringent wash 6 mL 12 mL Elution Elute the RNA with Buffer R4 preheating to 50 C improves the yield of RNA but may increase DNA c
36. uL 40 400 uL Add Buffer W1 and proceed with step 2 400 pL 1 6 mL B For solid samples e g RNA pellets Add Buffer W5 Dissolve the pellet very carefully if necessary by incubation at 65 C for 1 3 min 1 2 mL 7 5 mL Add Buffer W6 mix and proceed with the centrifugation of step 2 400 uL 2 5 mL C For low salt RNA solutions e g run off transcripts pre purified RNA Add 1 5 volume Buffer R3 and proceed with step 3 2 Adjustment of binding conditions Add Buffer W3 mix the sample and incubate for 5 min at room temperature 50 uL 200 pL Add Buffer RO to the supernatant and mix carefully 5 mL 18 mL If necessary centrifuge the solution 10 min 12 000 x g 4 C in order to remove insoluble particles and to avoid clogging of the column and collect the clear supernatant MACHEREY NAGEL 03 2013 Rev 06 27 NucleoBond RNA DNA AXR 80 AXR 400 3 Equilibration Equilibrate a NucleoBond AXR Column with Buffer R1 1mL 3 mL 4 Binding Transfer the clear supernatant to the column 5 Wash Wash the NucleoBond AXR Column with Buffer R1 for purification of tRNA or tRNA mRNA rRNA or Buffer R1 2 1 1 for purification of rPRNA mRNA or Buffer R2 for purification of rRNA or viral RNA stringent wash 6 Elution Elute the RNA with Buffer R3 preheated to 50 C 3mL 6mL 7 Precip
37. ube Add the Proteinase K stock solution 20 mg mL to the homogenate Mix well by vortexing for 30 s 25 uL 100 uL 450 pL Incubate the sample at 50 C for 2 hours If the lysate is not clear after incubation with Proteinase K the incubation time should be prolonged If any insoluble cell components are observed the sample should be clarified by a short centrifugation 5 000 x g 5 min Note It is very important to obtain a clear lysate in order to avoid clogging of the column MACHEREY NAGEL 03 2013 Rev 06 37 NucleoBond AXG and NucleoBond Buffer Set AXG 20 AXG 100 AXG 500 2 Equilibration Equilibrate the column with Buffer N2 1mL 2mL 5 mL 3 Binding Add Buffer N2 room temperature to the sample Vortex the mixture for 15 s at maximum speed Load the sample onto the column Allow it to enter the resin by gravity flow 4 Wash Wash the column with Buffer N3 3x1mL 3x4mL 5 Elution Elute the genomic DNA with Buffer N5 A second elution step with the same volume will increase the yield slightly 10 15 1mL 5mL 8 mL 6 Precipitation Add 0 7 volume of isopropanol room temperature mix incubate 30 60 min at room temperature and centrifuge at 4 C 15 000 rpm for 25 min 700 uL 3 5 mL 5 6 mL If the pellet looks glassy air dry it not longer than 15 min and redissolve it in slightly alkaline buffer e g TE pH 8 overnigh
38. ut or eluted with NucleoBond RNA DNA using Buffers R1 R4 which contain increasing amounts of KCI at pH 6 3 Table 2 Finally high molecular weight DNA can be eluted after efficient washing of the column at a slightly alkaline pH The RNA or DNA is then precipitated to remove the salt and dissolved in TE buffer or water for further use Table 2 Elution conditions for different RNA species Compound KCI salt Wash with Elute with concentration for elution tRNA 0 45 0 65 M Buffer R1 Buffer R2 R4 5S rRNA 0 65 0 85 M Buffer R1 R2 1 1 Buffer R3 R4 mRNA 0 70 1 15 M Buffer R1 R2 1 1 Buffer R3 R4 rRNA 0 95 1 10 M Buffer R1 R2 1 1 Buffer R3 R4 Buffer R2 tRNA 5S rRNA 0 45 1 15 M Buffer R1 Buffer R3 R4 mRNA rRNA 3 2 Kit specifications NucleoBond RNA DNA purification kits contain NucleoBond AXR Columns and appropriate buffers to purify high molecular weight RNA and DNA from eukaryotic cells bacteria tissue yeast liquid samples and reaction mixtures Kits are available with two column sizes AXR 80 and 400 for 80 ug NucleoBond RNA DNA 80 and 400 ug RNA NucleoBond RNA DNA 400 NucleoBond CB purification kits contain NucleoBond AXG Colums and appropriate buffers to purify high molecular weight genomic DNA from cell cultures and blood Kits are available with three column sizes AXG 20 100 and 500 for 20 ug NucleoBond CB 20 100 ug NucleoBond CB 100 and 500 ug DNA NucleoBond CB 500
39. with Proteinase K the incubation time should be prolonged If any insoluble cell components are observed the sample should be clarified by a short centrifugation 5 000 x g 5 min Note It is very important to obtain a clear lysate in order to avoid clogging of the column 2 Equilibration Equilibrate the column with Buffer N2 1mL 2mL 5 mL 3 Binding Add Buffer N2 room temperature to the sample Vortex the mixture for 15 s at maximum speed Load the sample onto the column Allow it to enter the resin by gravity flow 4 Wash Wash the column with Buffer N3 3x1mL 3x4mL 32 MACHEREY NAGEL 03 2013 Rev 06 NucleoBond AXG and NucleoBond Buffer Set AXG 20 AXG 100 AXG 500 Elution Elute the genomic DNA with Buffer N5 A second elution step with the same volume will increase the yield slightly 15 20 1mL 5 mL 8 mL Precipitation Add 0 7 volume of isopropanol room temperature mix incubate 30 60 min at room temperature and centrifuge at 4 C 15 000 rpm for 25 min If the pellet looks glassy air dry it not longer than 15 min and redissolve it in slightly alkaline buffer e g TE pH 8 overnight on a shaker or at 55 C for 1 2 hours If a white pellet is obtained additionally wash it with 70 ethanol and redissolve it as described above MACHEREY NAGEL 03 2013 Rev 06 33 NucleoBond AXG and NucleoBond Buffer Set 8 2
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