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 manualNucleoBond PC 20 NucleoBond PC 100 NucleoBond PC 500 NucleoBond BAC 100 NucleoBond PC 2000 NucleoBond PC 10000 May 2018 / Rev.

2 11 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 DNA purification (Mini, Midi, Maxi, Mega, Giga)Protocol at a glance (Rev. 11)Mini (AX 20) Midi (AX 100) Maxi (AX 500)Mega(AX 2000) Giga (AX 10000)1 Cultivate and harvest bacterial cells4,500 6,000 x g 4 C, 15 min4,500 6,000 x g 4 C, 15 min4,500 6,000 x g 4 C, 15 min4,500 6,000 x g 4 C, 15 min4,500 6,000 x g 4 C, 15 min2 Cell lysisHigh copy / low-copyBuffer mL / mL4 mL / 8 mL12 mL / 24 mL45 mL / 90 mL120 mL / Buffer mL / mL RT, < 5 min4 mL / 8 mL RT, < 5 min12 mL / 24 mL RT, < 5 min45 mL / 90 mL RT, < 5 min120 mL / RT, < 5 minBuffer mL / mL 0 C, 5 min4 mL / 8 mL 0 C, 5 min12 mL / 24 mL 0 C, 5 min45 mL / 90 mL 0 C, 5 min120 mL / 0 C.

3 5 min3 Equilibration of the columnBuffer N2 1 mLBuffer N2 mLBuffer N2 6 mLBuffer N2 20 mLBuffer N2 100 mL4 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 min5 BindingLoad cleared lysate onto the columnLoad cleared lysate onto the columnLoad cleared lysate onto the columnLoad cleared lysate onto the columnLoad cleared lysate onto the column6 WashingBuffer N3 High copy 2 x mLLow copy 2 x 2 mLBuffer N3 High copy 10 mLLow copy 12 mLBuffer N3 High copy 32 mLLow copy 2 x 18 mLBuffer N3 High copy 2 x 35 mLLow copy 2 x 50 mLBuffer N3 High copy 2 x 100 mL 7 ElutionBuffer N5 1 mLBuffer N5 5 mLBuffer N5 15 mLBuffer N5 25 mLBuffer N5 100 mL8 PrecipitationIsopropanol mLIsopropanol mLIsopropanol 11 mLIsopropanol 18 mLIsopropanol 70 mL 4,500 x g 4 C, 30 min 4,500 x g 4 C.

4 30 min 4,500 x g 4 C, 30 min 4,500 x g 4 C, 30 min 4,500 x g 4 C, 30 min9 Wash and dry DNA pellet70 % ethanol 500 L70 % ethanol 2 mL70 % ethanol 5 mL70 % ethanol 7 mL70 % ethanol 10 mL 4,500 x g RT, 10 min 4,500 x g RT, 10 min 4,500 x g RT, 10 min 4,500 x g RT, 10 min 4,500 x g RT, 10 min5 10 min5 10 min10 20 min30 60 min30 60 min10 Reconstitute DNAA ppropriate volume of TEAppropriate volume of TEAppropriate volume of TEAppropriate volume of TEAppropriate volume of TE3 Macherey-Nagel 05/2018, Rev. 11 Plasmid DNA PurificationTable of contents1 Components Kit contents Reagents and equipment to be supplied by user 92 About this user manual 103 Product description The basic principle Kit specifications High- / low-copy Plasmid purification Filtration of the lysate Elution procedure 144 Storage conditions and preparation of working solutions 155 Safety instructions 166 Optimization of the Plasmid isolation procedure Estimation of optimal culture volume SDS precipitation in Buffer S2 207 NucleoBond Plasmid purification General procedure High-copy Plasmid purification (Mini, Midi, Maxi)

5 High-copy Plasmid purification (Mega, Giga) Low-copy Plasmid purification (Mini, Midi) Low-copy Plasmid purification (Maxi / BAC, Mega) 308 Appendix Determination of DNA yield and quality Troubleshooting Ordering information References Product use restriction / warranty 41 Macherey-Nagel 05/2018, Rev. 114 Plasmid DNA Purification1 Components Kit contentsNucleoBond PC 20 REF20 preps740571100 Buffer S125 mL100 mLLysis Buffer S225 mL100 mLNeutralization Buffer S325 mL100 mLEquilibration Buffer N225 mL125 mLWash Buffer N3125 mL3 x 125 mLElution Buffer N532 mL125 mLRNase A (lyophilized)* mg10 mgNucleoBond AX 20 Columns 20100 Plastic Washers1010 User Manual11* For preparation of working solutions and storage conditions see section 05/2018, Rev.

6 11 Plasmid DNA Kit contents continuedNucleoBond PC 100 REF20 preps740573100 Buffer S1100 mL500 mLLysis Buffer S2100 mL500 mLNeutralization Buffer S3100 mL500 mLEquilibration Buffer N270 mL500 mLWash Buffer N32 x 125 mL1000 mL 125 mLElution Buffer N5125 mL1000 mLRNase A (lyophilized)*10 mg50 mgNucleoBond AX 100 Columns 20100 NucleoBond Folded Filters20100 Plastic Washers1010 User Manual11* For preparation of working solutions and storage conditions see section 05/2018, Rev. 116 Plasmid DNA Kit contents continuedNucleoBond PC 500 REF10 preps74057425 Buffer S1150 mL500 mL750 mL2 x 750 mLLysis Buffer S2150 mL500 mL750 mL2 x 750 mLNeutralization Buffer S3150 mL500 mL750 mL2 x 750 mLEquilibration Buffer N270 mL200 mL500 mL1000 mLWash Buffer N32 x 250 mL1000 mL2 x 1000 mL3 x 1000 mL 500 mLElution Buffer N5200 mL500 mL1000 mL2 x 1000 mLRNase A (lyophilized)*15 mg50 mg75 mg2 x 75 mgNucleoBond AX 500 Columns 102550100 NucleoBond Folded Filters XL102550100 Plastic Washers5101010 User Manual1111* For preparation of working solutions and storage conditions see section 05/2018, Rev.

7 11 Plasmid DNA Kit contents continuedNucleoBond PC 2000 NucleoBond PC 10000 REF5 preps7405765 preps740593 Resuspension Buffer S1250 mL750 mLLysis Buffer S2250 mL750 mLNeutralization Buffer S3250 mL750 mLEquilibration Buffer N2125 mL500 mL 125 mLWash Buffer N32 x 250 mL1000 mL 125 mLElution Buffer N5200 mL500 mL 125 mLRNase A (lyophilized)*25 mg75 mgNucleoBond AX 2000 Columns 5 NucleoBond AX 10000 Columns 5 NucleoBond Folded Filters XL5 NucleoBond Folded Filters Type 1 10 NucleoBond Folded Filters Type 2 10 Plastic Washers5 User Manual11* For preparation of working solutions and storage conditions see section 05/2018, Rev. 118 Plasmid DNA Kit contents continuedNucleoBond BAC 100 REF10 preps740579 Resuspension Buffer S1250 mLLysis Buffer S2250 mLNeutralization Buffer S3250 mLEquilibration Buffer N270 mLWash Buffer N32 x 250 mLElution Buffer N5200 mLRNase A (lyophilized)*25 mgNucleoBond BAC 100 Columns 10 NucleoBond Folded Filters XL10 Plastic Washers5 User Manual1* For preparation of working solutions and storage conditions see section 05/2018, Rev.

8 11 Plasmid DNA Reagents and equipment to be supplied by userReagents Isopropanol (room-temperatured) 70 % ethanol (room-temperatured) Ice Buffer for reconstitution of DNA ( , TE buffer or sterile H2O)Equipment Standard microbiological equipment for growing and harvesting bacteria ( , 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 5000 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 protocolMACHEREY-NAGEL 05/2018, Rev.

9 1110 Plasmid DNA Purification2 About this user manualIt 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 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 High-copy Plasmid purification ):Mini (AX 20) Midi (AX 100) Maxi (AX 500)1 Cell lysisCarefully resuspend the pellet of bacterial cells in Buffer S1 + RNase 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.

10 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 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 05/2018, Rev. 11 Plasmid DNA Purification3 Product The basic principleNucleoBond PC / BAC kits employ a modified alkaline /SDS lysis procedure to prepare the bacterial cell pellet for Plasmid purification .