Plasmid DNA purification - Macherey-Nagel AG

1 US:Tel.: +1 484 821 0984 Fax: +1 484 821 1272 E-mail: GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren / International:Tel.: +49 24 21 969-0 Fax: +49 24 21 969-199 E-mail: +41 62 388 55 00 Fax: +41 62 388 55 05 E-mail: +33 388 68 22 68 Fax: +33 388 51 76 88 E-mail: GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren / International:Tel.: +49 24 21 969-0 Fax: +49 24 21 969-199 E-Mail: +41 62 388 55 00 Fax: +41 62 388 55 05 E-Mail: +33 388 68 22 68 Fax: +33 388 51 76 88 E-Mail: ISO 9001 ZERTIFIZIERTMACHEREY-NAGEL GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren / International:Tel.: +49 24 21 969-0 Fax: +49 24 21 969-199 E-Mail: +41 62 388 55 00 Fax: +41 62 388 55 05 E-Mail: +33 388 68 22 68 Fax: +33 388 51 76 88 E-Mail: ISO 9001 ZERTIFIZIERTP lasmid DNA purificationUser manualNucleoSpin Plasmid NucleoSpin Plasmid (NoLid) NucleoSpin Plasmid QuickPureDecember 2017 / Rev.

2 10 Plasmid DNA purificationProtocol-at-a-glance (Rev. 10) Macherey-Nagel GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren GermanyTel.: +49 24 21 969-270 Fax: +49 24 21 969-199 PlasmidNucleoSpin Plasmid (NoLid)NucleoSpin Plasmid QuickPure1 Cultivate and harvest bacterial cells11,000 x g, 30 s11,000 x g, 30 s2 Cell lysis250 L Buffer A1250 L Buffer A1250 L Buffer A2RT, up to 5 min250 L Buffer A2RT, up to 5 min300 L Buffer A3300 L Buffer A33 Clarification of the lysate11,000 x g, 5 10 min11,000 x g, 5 min4 Bind DNALoad supernatantLoad supernatant11,000 x g, 1 min11,000 x g, 1 min5 Wash silica membrane(Optional: 500 L Buffer AW: RT or 50 C)450 L Buffer AQ600 L Buffer A411,000 x g, 1 min11,000 x g, 3 min6 Dry silica membraneDrying is performed during centrifugation of the single washing step11,000 x g, 2 min7 Elute DNA50 L Buffer AERT, 1 min50 L Buffer AERT, 1 min11,000 x g, 1 min11,000 x g, 1 min3 Macherey-Nagel 12/2017, Rev.

3 10 Plasmid DNA purificationTable of contents1 Components Kit contents Reagents, consumables, and equipment to be supplied by user About this user manual 72 Product description Basic principle Kit specifications Growth of bacterial cultures Lysate neutralization and LyseControl Elution procedures 113 Storage conditions and preparation of working solutions 124 Safety instructions 135 NucleoSpin Plasmid / Plasmid (NoLid) protocols Isolation of high-copy Plasmid DNA from E. coli Isolation of low-copy plasmids, P1 constructs, or cosmids 176 NucleoSpin Plasmid QuickPure protocol isolation of high-copy Plasmid DNA from E. coli 197 NucleoSpin Plasmid / Plasmid (NoLid), and NucleoSpin Plasmid QuickPure protocols Plasmid DNA clean up 228 Appendix Troubleshooting Ordering information References Product use restriction / warranty 26 Macherey-Nagel 12/2017, Rev.

4 104 Plasmid DNA purification1 Kit contentsNucleoSpin PlasmidREF10 preps preps preps Buffer A15 mL15 mL75 mLLysis Buffer A25 mL15 mL100 mLNeutralization Buffer A35 mL20 mL100 mLWash Buffer AW6 mL30 mL2 x 75 mLWash Buffer A4 (Concentrate)*6 mL12 mL2 x 25 mLElution Buffer AE**13 mL13 mL60 mLRNase A (lyophilized)* mg6 mg30 mgNucleoSpin Plasmid Columns (white rings)1050250 Collection Tubes (2 mL)1050250 user manual111* For preparation of working solutions and storage conditions see section 3.** Composition of Elution Buffer AE: 5 mM Tris/HCl, pH 12/2017, Rev. 10 Plasmid DNA purificationKit contents continuedNucleoSpin Plasmid (NoLid)REF10 preps preps preps Buffer A15 mL15 mL75 mLLysis Buffer A25 mL15 mL100 mLNeutralization Buffer A35 mL20 mL100 mLWash Buffer AW6 mL30 mL2 x 75 mLWash Buffer A4 (Concentrate)*6 mL12 mL2 x 25 mLElution Buffer AE**13 mL13 mL60 mLRNase A (lyophilized)* mg6 mg30 mgNucleoSpin Plasmid (NoLid) Columns (white rings)1050250 Collection Tubes (2 mL)1050250 user manual111* For preparation of working solutions and storage conditions see section 3.

5 ** Composition of Elution Buffer AE: 5 mM Tris/HCl, pH 12/2017, Rev. 106 Plasmid DNA purificationKit contents continuedNucleoSpin Plasmid QuickPureREF10 preps preps preps Buffer A15 mL15 mL75 mLLysis Buffer A2 (without LyseControl)5 mL15 mL100 mLNeutralization Buffer A35 mL20 mL100 mLWash Buffer AQ (Concentrate)* 6 mL6 mL25 mLElution Buffer AE**13 mL13 mL60 mLRNase A (lyophilized)* mg6 mg30 mgNucleoSpin Plasmid QuickPure Columns (dark yellow rings)1050250 Collection Tubes (2 mL)1050250 user manual111* For preparation of working solutions and storage conditions see section 3.** Composition of Elution Buffer AE: 5 mM Tris/HCl, pH 12/2017, Rev. 10 Plasmid DNA Reagents, consumables, and equipment to be supplied by userReagents 96 100 % ethanolConsumables mL microcentrifuge tubes for sample lysis and DNA elution Disposable pipette tipsEquipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Heating-block (NucleoSpin Plasmid / Plasmid (NoLid): for large constructs or optional Wash Buffer AW) Personal protection equipment (lab coat, gloves, goggles) About this user manualIt is strongly recommended for first time users to read the detailed protocol sections of the user manual NucleoSpin Plasmid / Plasmid (NoLid) or NucleoSpin Plasmid QuickPure before using these products.

6 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 Please visit the Macherey-Nagel website to verify that you are using the latest revision of this user contact Technical Service regarding information about changes to the current user manual compared with previous 12/2017, Rev. 108 Plasmid DNA purification2 Product Basic principleWith the NucleoSpin Plasmid method, the pelleted bacteria are resuspended (Buffer A1) and Plasmid DNA is liberated from the E. coli host cells by SDS / alkaline lysis (Buffer A2). Buffer A3 neutralizes the resulting lysate and creates appropriate conditions for binding of Plasmid DNA to the silica membrane of the NucleoSpin Plasmid / Plasmid (NoLid) or NucleoSpin Plasmid QuickPure Column.

7 Precipita-ted protein, genomic DNA, and cell debris are then pelleted by a centrifugation step. The supernatant is loaded onto a NucleoSpin Plasmid / Plasmid (NoLid) or NucleoSpin Plasmid QuickPure Column. With the NucleoSpin Plasmid / Plasmid (NoLid) kit contaminations like salts, metabolites, and soluble macromolecular cellular components are removed by simple washing with ethanolic Buffer A4. Pure Plasmid DNA is finally eluted under low ionic strength conditions with slightly alkaline Buffer AE (5 mM Tris / HCl, pH ). If host strains with high levels of nucleases are used, an additional washing step with preheated Buffer AW is recommended. Additional washing with Buffer AW will also increase the reading length of automated fluorescent DNA sequencing the NucleoSpin Plasmid QuickPure kit contaminations like salts, metabolites, nucleases, and soluble macromolecular cellular components are removed by only a single washing step with Buffer AQ.

8 Pure Plasmid DNA is finally eluted under low ionic strength conditions with slightly alkaline Buffer AE (5 mM Tris/HCl, pH ). Kit specifications The NucleoSpin Plasmid / Plasmid (NoLid) and NucleoSpin Plasmid QuickPure kits are designed for the rapid, small-scale preparation of highly pure Plasmid DNA (mini preps). The NucleoSpin Plasmid / Plasmid (NoLid) Columns offer a very high DNA binding capacity of up to 60 g. This, however, requires thorough washing. Therefore, the kit includes an additional Wash Buffer AW which is strongly recommended for host strains with high levels of endonucleases like ABLE, HB101, or JM110. The NucleoSpin Plasmid QuickPure Column features a new specially treated silica membrane which allows speeding up the procedure by a combined washing and drying step. No additional steps are necessary if nuclease rich host strains are used.

9 The number of washing and drying steps is reduced from 3 to only 1! Therefore, the hands-on time is less than 11 min. However, the DNA binding capacity is limited to 15 g. The Plasmid DNA prepared with both kits, NucleoSpin Plasmid / Plasmid (NoLid) and NucleoSpin Plasmid QuickPure, is suitable for applications like automated fluorescent DNA sequencing, PCR, or any kind of enzymatic manipulation. 9 Macherey-Nagel 12/2017, Rev. 10 Plasmid DNA purification Furthermore, support protocols allow purification of low-copy plasmids from larger culture volumes, purification of plasmids from Gram positive bacteria, and clean up of plasmids from reaction 1: Kit specifications at a glanceParameterNucleoSpin Plasmid / Plasmid (NoLid)NucleoSpin Plasmid QuickPureCulture volume1 5 mL high copy 5 10 mL low copy1 3 mL high copyTypical yield< 25 g (1 5 mL culture) < 40 g (5 10 mL culture)< 15 g (1 3 mL culture)Elution volume50 L50 LBinding capacity60 g15 gVectors< 50 kbp< 15 kbpPreparation time*25 min /18 preps11 min /18 prepsFormatMini spin columnMini spin Growth of bacterial culturesYield and quality of Plasmid DNA highly depend on the type of culture media and antibiotics, the bacterial host strain, the Plasmid type, size, or copy number.

10 For cultivation of bacterial cells harbouring standard high-copy plasmids, we recommend LB (Luria Bertani) medium. The cell culture should be incubated at 37 C with constant shaking (200 250 rpm) preferably 12 16 h over night. Usually an OD of 3 6 can be achieved. Alternatively, rich media like 2 x YT (Yeast / Tryptone), TB (Terrific Broth), or CircleGrow can be used. In this case bacteria grow faster, reach the stationary phase much earlier than in LB medium ( 12 h), and higher cell masses can be reached. However, this does not necessarily yield more Plasmid DNA. Overgrowing a culture might lead to a higher percentage of dead or starving cells and the resulting Plasmid DNA might be partially degraded or contaminated with chromosomal DNA. To find the optimal culture conditions, the culture medium and incubation times have to be optimized for each host strain / Plasmid construct combination cultures should be grown under antibiotic selection at all times to ensure Plasmid propagation.