nucleobond plasmid purification user manual

MACHEREY-NAGEL www.mn-net.com MACHEREY-NAGEL MACHEREY-NAGEL EN ISO 9001: 2008 CERTIFIED MN A0 25 74 4 R0 9e n1 /4 /0 /11 .11 Pr int ed in G er m an y Plasmid DNA purification User manual NucleoBond® PC 20 NucleoBond® PC 100 NucleoBond® PC 500 NucleoBond® BAC 100 NucleoBond® PC 2000 NucleoBond® PC 10000 November 2011 / Rev. 09 MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · Germany Tel.: +49 24 21 969-270 · Fax: +49 24 21 969-199 · tech-bio@mn-net.com · www.mn-net.com Plasmid DNA Purification (Mini, Midi, Maxi, Mega, Giga) Protocol-at-a-glance (Rev. 09) Mini (AX 20) Midi (AX 100) Maxi (AX 500) Mega (AX 2000) Giga (AX 10000) 1 Cultivate and harvest bacterial cells 4,500–6,000 x g 4 °C, 15 min 4,500–6,000 x g 4 °C, 15 min 4,500–6,000 x g 4 °C, 15 min 4,500–6,000 x g 4 °C, 15 min 4,500–6,000 x g 4 °C, 15 min 2 Cell lysis High copy / low-copy Buffer S1 0.4 mL / 0.8 mL 4 mL / 8 mL 12 mL / 24 mL 45 mL / 90 mL 120 mL / – Buffer S2 0.4 mL / 0.8 mL RT, < 5 min 4 mL / 8 mL RT, < 5 min 12 mL / 24 mL RT, < 5 min 45 mL / 90 mL RT, < 5 min 120 mL / – RT, < 5 min Buffer S3 0.4 mL / 0.8 mL 0 °C, 5 min 4 mL / 8 mL 0 °C, 5 min 12 mL / 24 mL 0 °C, 5 min 45 mL / 90 mL 0 °C, 5 min 120 mL / – 0 °C, 5 min 3 Equilibration of the column Buffer N2 1 mL Buffer N2 2.5 mL Buffer N2 6 mL Buffer N2 20 mL Buffer N2 100 mL 4 Clarification of the lysate Centrifugation 12,000 x g 15 min Folded Filter or centrifugation 12,000 x g 25 min Folded Filter or centrifugation 12,000 x g 40 min Folded Filter or centrifugation 12,000 x g 50 min Folded Filter or centrifugation 12,000 x g 60 min 5 Binding Load cleared lysate onto the column Load cleared lysate onto the column Load cleared lysate onto the column Load cleared lysate onto the column Load cleared lysate onto the column 6 Washing Buffer N3 High copy 2 x 1.5 mL Low copy 2 x 2 mL Buffer N3 High copy 10 mL Low copy 12 mL Buffer N3 High copy 32 mL Low copy 2 x 18 mL Buffer N3 High copy 2 x 35 mL Low copy 2 x 50 mL Buffer N3 High copy 2 x 100 mL 7 Elution Buffer N5 1 mL Buffer N5 5 mL Buffer N5 15 mL Buffer N5 25 mL Buffer N5 100 mL 8 Precipitation Isopropanol 0.75 mL Isopropanol 3.5 mL Isopropanol 11 mL Isopropanol 18 mL Isopropanol 70 mL ≥ 15,000 x g 4 °C, 30 min ≥ 15,000 x g 4 °C, 30 min ≥ 15,000 x g 4 °C, 30 min ≥ 15,000 x g 4 °C, 30 min ≥ 15,000 x g 4 °C, 30 min 9 Wash and dry DNA pellet 70 % ethanol 500 μL 70 % ethanol 2 mL 70 % ethanol 5 mL 70 % ethanol 7 mL 70 % ethanol 10 mL ≥ 15,000 x g RT, 10 min ≥ 15,000 x g RT, 10 min ≥ 15,000 x g RT, 10 min ≥ 15,000 x g RT, 10 min ≥ 15,000 x g RT, 10 min 5–10 min 5–10 min 10–20 min 30–60 min 30–60 min 10 Reconstitute DNA Appropriate volume of TE Appropriate volume of TE Appropriate volume of TE Appropriate volume of TE Appropriate volume of TE 3 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 Table of contents 1 Components 5 1.1 Kit contents 5 1.2 Reagents and equipment to be supplied by user 10 2 Introduction 11 2.1 Properties 11 2.2 About this user manual 12 3 Product description 13 3.1 The basic principle 13 3.2 Kit specifications 13 3.3 Buffer compositions 14 3.4 High- / low-copy plasmid purification 15 3.5 Filtration of the lysate 16 3.6 Elution procedure 17 4 Storage conditions and preparation of working solutions 18 5 Safety instructions 19 5.1 Risk and safety phrases 19 5.2 GHS classification 20 6 Growing of bacterial cultures 22 6.1 General considerations 22 6.2 Selection of culture media 23 6.3 Difficult-to-lyse strains 23 6.4 Chloramphenicol amplification of low-copy plasmids 23 7 NucleoBond® plasmid purification 24 7.1 General procedure 24 7.2 High-copy plasmid purification (Mini, Midi, Maxi) 24 7.3 High-copy plasmid purification (Mega, Giga) 27 7.4 Low-copy plasmid purification (Mini, Midi) 30 7.5 Low-copy plasmid purification (Maxi / BAC, Mega) 33 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 094 8 Appendix 36 8.1 Determination of DNA yield and quality 36 8.2 Troubleshooting 36 8.3 Ordering information 43 8.4 References 44 8.5 Product use restriction / warranty 45 5 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 1 Components 1.1 Kit contents NucleoBond® PC 20 20 preps 100 preps REF 740571 740571.100 Resuspension Buffer S1 25 mL 2 x 25 mL Lysis Buffer S2 25 mL 2 x 25 mL Neutralization Buffer S3 25 mL 2 x 25 mL Equilibration Buffer N2 25 mL 125 mL Wash Buffer N3 3 x 30 mL 3 x 125 mL Elution Buffer N5 32 mL 120 mL RNase A (lyophilized)* 2.5 mg 2 x 2.5 mg NucleoBond® AX 20 Columns  20 100 Plastic Washers 10 10 User Manual 1 1 * For preparation of working solutions and storage conditions see section 4. Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 096 1.1 Kit contents continued NucleoBond® PC 100 20 preps 100 preps REF 740573 740573.100 Resuspension Buffer S1 100 mL 500 mL Lysis Buffer S2 4 x 25 mL 500 mL Neutralization Buffer S3 100 mL 500 mL Equilibration Buffer N2 70 mL 2 x 150 mL Wash Buffer N3 250 mL 1000 mL 125 mL Elution Buffer N5 120 mL 3 x 200 mL RNase A (lyophilized)* 10 mg 50 mg NucleoBond® AX 100 Columns  20 100 NucleoBond® Folded Filters 20 100 Plastic Washers 10 10 User Manual 1 1 * For preparation of working solutions and storage conditions see section 4. 7 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 1.1 Kit contents continued NucleoBond® PC 500 10 preps 25 preps 50 preps 100 preps REF 740574 740574.25 740574.50 740574.100 Resuspension Buffer S1 150 mL 2 x 200 mL 2 x 400 mL 3 x 500 mL Lysis Buffer S2 150 mL 400 mL 2 x 400 mL 3 x 500 mL Neutralization Buffer S3 150 mL 400 mL 2 x 400 mL 3 x 500 mL Equilibration Buffer N2 70 mL 200 mL 2 x 200 mL 500 mL 200 mL Wash Buffer N3 2 x 250 mL 1000 mL 2 x 1000 mL 3 x 1000 mL 500 mL Elution Buffer N5 200 mL 500 mL 2 x 500 mL 3 x 500 mL 200 mL RNase A (lyophilized)* 15 mg 2 x 25 mg 2 x 40 mg 3 x 50 mg NucleoBond® AX 500 Columns  10 25 50 100 NucleoBond® Folded Filters XL 10 25 50 100 Plastic Washers 5 10 10 10 User Manual 1 1 1 1 * For preparation of working solutions and storage conditions see section 4. Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 098 1.1 Kit contents continued NucleoBond® PC 2000 NucleoBond® PC 10000 5 preps 5 preps REF 740576 740593 Resuspension Buffer S1 250 mL 750 mL Lysis Buffer S2 250 mL 750 mL Neutralization Buffer S3 250 mL 750 mL Equilibration Buffer N2 125 mL 500 mL 125 mL Wash Buffer N3 2 x 250 mL 1000 mL 125 mL Elution Buffer N5 200 mL 500 mL 120 mL RNase A (lyophilized)* 25 mg 80 mg NucleoBond® AX 2000 Columns  5 – NucleoBond® AX 10000 Columns  – 5 NucleoBond® Folded Filters XL 5 – NucleoBond® Folded Filters Type 1 – 10 NucleoBond® Folded Filters Type 2 – 10 Plastic Washers 5 – User Manual 1 1 * For preparation of working solutions and storage conditions see section 4. 9 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 1.1 Kit contents continued NucleoBond® BAC 100 10 preps REF 740579 Resuspension Buffer S1 2 x 150 mL Lysis Buffer S2 2 x 150 mL Neutralization Buffer S3 2 x 150 mL Equilibration Buffer N2 70 mL Wash Buffer N3 2 x 250 mL Elution Buffer N5 150 mL RNase A (lyophilized)* 2 x 15 mg NucleoBond® BAC 100 Columns  10 NucleoBond® Folded Filters XL 10 Plastic Washers 5 User Manual 1 * For preparation of working solutions and storage conditions see section 4. Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 0910 1.2 Reagents and equipment to be supplied by user Reagents • Isopropanol (room-temperatured) • 70 % ethanol (room-temperatured) • Ice • Buffer for reconstitution of DNA (e.g., TE buffer or sterile H2O) Equipment • Standard microbiological equipment for growing and harvesting bacteria (e.g., inoculating loop, culture tubes and flasks, 37 °C shaking incubator, and centrifuge with rotor and tubes or bottles for harvesting cells) • Funnels to hold the NucleoBond® Folded Filters for lysate filtration (NucleoBond® PC 100, 500, 2000, 10000, BAC 100) • NucleoBond® Rack (see ordering information) or equivalent holder • Refrigerated centrifuge capable of reaching ≥ 15,000 x g with rotor for the appropriate centrifuge tubes or bottles • Centrifugation tubes or vessels with suitable capacity for the volumes specified in the respective protocol 11 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 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 plasmid 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, carbohydrates, 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. 0 0.5 1 1.5 Salt concentration for elution [M (KCl)] Ab so rb an ce a t 2 60 n m Co m po un d cla ss Single-stranded DNA,Double-stranded DNA mRNA, 16S/23S rRNA 5S rRNA tRNA Proteins, dyes, polysaccharides, metabolites, trinucleotides tR NA rR NA Pl as m id D NA , la rg e co ns tru ct s Plasmid DNA, large constructs 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. Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 0912 2.2 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoBond® Plasmid 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. The protocols in this manual are organized as follows: The volumes of the respective buffers used for a particular column size are high-lighted. Each procedural step is arranged like the following example (taken from section 7.2 High-copy plasmid purification): Mini (AX 20) Midi (AX 100) Maxi (AX 500) 1 Carefully resuspend the pellet of bacterial cells in Buffer S1 + RNase A. Please see section 6.3 regarding difficult-to-lyse strains. 0.4 mL 4 mL 12 mL For example, if you are performing a Mini prep to purify plasmid DNA you will find volumes or incubation times in the white boxes. 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 pellet of the bacterial cells has to be resuspended in 0.4 mL of Buffer S1 when performing a Mini prep using NucleoBond® AX 20 Columns, in 4 mL of Buffer S1 when performing a Midi prep using NucleoBond® AX 100 Columns, and in 12 mL of Buffer S1 when performing a Maxi prep using NucleoBond® AX 500 Columns. 13 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 3 Product description 3.1 The basic principle NucleoBond® PC / BAC kits employ a modified alkaline /SDS lysis procedure to prepare the bacterial cell pellet for plasmid purification. Both chromosomal and plasmid DNA are denatured under these alkaline conditions. Potassium acetate is then added to the denatured lysate, which causes the formation of a precipitate containing chromosomal DNA and other cellular compounds. The potassium acetate buffer also neutralizes the lysate. Plasmid DNA can revert to its native supercoiled structure and remains in solution. After equilibrating the appropriate NucleoBond® Column with equilibration buffer, plasmid DNA is bound to the anion-exchange resin and finally eluted after efficient washing of the column. After precipitation of the eluted DNA it can easily be dissolved in TE buffer for further use. 3.2 Kit specifications • NucleoBond® Plasmid purification kits contain NucleoBond® Columns, appropriate buffers, and RNase A. Kits are available for each column size: Mini (PC 20), Midi (PC 100), Maxi (PC 500, BAC 100), Mega (PC 2000), and Giga (PC 10000). • The protocols are suitable for purifying most plasmids ranging from 3–10 kbp, cosmids from 10–50 kbp, and very large constructs (P1 constructs, BACs, PACs) up to 300 kbp. • NucleoBond® 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 1). Table 1: NucleoBond® Column binding capacities NucleoBond® Column Binding capacity AX 20 20 μg AX 100 100 μg AX 500 500 μg BAC 100 100 μg AX 2000 2 mg AX 10000 10 mg • All NucleoBond® Columns are resistant to organic solvents such as alcohol, chloroform, and phenol and are free of DNase and RNase. Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 0914 • 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 Buffer compositions Resuspension Buffer S1: • 50 mM Tris-HCl, 10 mM EDTA, 100 μg / mL RNase A, pH 8.0 Lysis Buffer S2: • 200 mM NaOH, 1 % SDS Neutralization Buffer S3: • 2.8 M KAc, pH 5.1 Equilibration Buffer N2: • 100 mM Tris, 15 % ethanol, 900 mM KCl, 0.15 % Triton X-100, adjusted to pH 6.3 with H3PO4 Wash Buffer N3: • 100 mM Tris, 15 % ethanol, 1.15 M KCl, adjusted to pH 6.3 with H3PO4 Elution Buffer N5: • 100 mM Tris, 15 % ethanol, 1 M KCl, adjusted to pH 8.5 with H3PO4 Note: Keep all buffers tightly capped. The concentration of KCl required for eluting the desired nucleic acid is highly dependent on the pH value of the eluent. For this reason, pH values must be carefully controlled if the buffers have been prepared by 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 H3PO4 or KOH. 15 Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 09 3.4 High- / low-copy plasmid purification NucleoBond® PC kits are recommended for the isolation of high-copy plasmids (> 20 copies / cell), however, low-copy plasmids (< 20 copies / cell) can be isolated as well. If you are purifying low-copy plasmids you will need to supplement the NucleoBond® PC kits with additional buffers. We recommend the NucleoBond® Buffer Set I (REF 740601) for routine purification of low-copy plasmids. The NucleoBond® Buffer Set I can be used in connection with NucleoBond® PC kits for the isolation of low-copy plasmids. In this combination it is sufficient for • NucleoBond® PC 500 kit (REF 740574), 10 preparations low-copy plasmid purification • NucleoBond® PC 100 kit (REF 740573), 20 preparations low-copy plasmid purification • NucleoBond® PC 20 kit (REF 740571.100), 100 preparations low-copy plasmid purification. In connection with NucleoBond® AX Columns the NucleoBond® Buffer Set I can be used for the isolation of high-copy plasmids. In this combination it is sufficient for • NucleoBond® PC 500 Columns (REF 740531) 5 preparations high-copy plasmid purification • NucleoBond® PC 100 Columns (REF 740521) 10 preparations high-copy plasmid purification • NucleoBond® PC 20 Columns (REF 740511) 50 preparations high-copy plasmid purification. The NucleoBond® BAC 100 kit is recommended for the isolation of low-copy plasmids and contains sufficient buffer to perform 10 maxi preps. The kit contains BAC 100 columns, which can bind up to 500 μg of plasmid DNA. Typically yields are 10–100 μg from 500 mL fermentation broth depending on copy number and size of constructs (also see section 6 for further information regarding the growing of bacterial cultures). The protocol for the isolation of low-copy plasmids using the NucleoBond® BAC 100 kit can be found in section 7.5. Plasmid DNA purification MACHEREY-NAGEL – 11 / 2011, Rev. 0916 3.5 Filtration of the lysate After alkaline lysis, the solution has to be cleared from precipitated protein and cell debris in order to prevent clogging of the NucleoBond® Columns. There are the following three options: 1) Use the NucleoBond® Folded Filters provided with the kits. The gentle filtration prevents shearing of plasmids and large constructs, such as cosmid, PAC, or BAC DNA. For NucleoBond® PC 100, 500, 2000, BAC 100 place one filter in a funnel of appropriate size. For NucleoBond® PC 10000 put a folded filter of type 1 into a folded filter of type 2 and place the combination in a funnel. The two types of filters differ in pore size to allow a fast and complete removal of large amounts of precipitate. Wet the filter(s) with a few drops of Equilibration Buffer N2 and load the bacterial lysate onto the wet filter(s). Either collect the flow-through in a separate vessel or position funnel and filter directly on top of the NucleoBond® Column to clear and load the lysate in one time-saving step (see Figure 2). Figure 2: Correct use of the NucleoBond® Folded Filter, NucleoBond® Column placed in a Plastic Washer 2) Use NucleoBond® Bottle Top Filters for a very fast and complete, vacuum assisted filtration. Two different types of filters can be ordered separately (see ordering information) for NucleoBond® PC 2000 (Type 1) or NucleoBond® PC 10000 (Type 2). Attach a bottle top filter to a suitable flask (e.g., Schott), load the bacterial lysate and apply the vacuum (see Figure 3). The lysate is cleared and sterile filtered within 3–5 minutes and can then be loaded onto the NucleoBond® Column. 3) Clearing the lysate by centrifugation is recommend- ed for either very small sample volumes (NucleoBond® PC 20) or very large volumes (low-copy protocol). Centrifuge the lysate for 5–30 minutes at > 12.000 x g at room temperature or better 4 °C. Apply the cleared lysate directly to the NucleoBond® Column or pass the lysate through a wet folded filter to remove remaining particles before loading the colum