Genomic DNA from tissue - mn-net.com

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 9001:2008 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 9001:2008 ZERTIFIZIERTG enomic DNA from tissueUser manualNucleoSpin tissue Januar 2017 / Rev.

2 17 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 from tissueProtocol-at-a-glance (Rev. 17)NucleoSpin Tissue1 Prepare sampleCut 25 mg into small pieces2 Pre-lyse sample180 L T125 L Proteinase K56 C, 1 3 h3 Lyse sample200 L B370 C, 10 min4 Adjust DNA binding conditions210 L 96 100 % ethanol5 Bind DNALoad all11,000 x g, 1 min6 Wash silica membrane1st wash2nd wash500 L BW600 L B51st and 2nd11,000 x g, 1 min7 Dry silica membrane11,000 x g, 1 min8 Elute highly pure DNA100 L BER T, 1 min11,000 x g, 1 min3 MACHEREY-NAGEL 01/2017, Rev.

3 17 Genomic DNA from tissueTable of contents1 Components Kit contents Reagents, consumables, and equipment to be supplied by user About this user manual 62 Product description The basic principle Kit specifications Elution procedures 83 Storage conditions and preparation of working solutions 104 Safety instructions 115 Standard protocol for human or animal tissue and cultured cells 136 Support protocols Support protocol for mouse or rat tails Support protocol for bacteria Support protocol for yeast Support protocol for dried blood spots ( , NucleoCards, FTA cards, Guthrie cards) Support protocol for Genomic DNA and viral DNA from blood samples Support protocol for hair roots Support protocol for paraffin-embedded tissue Support protocol for Genomic DNA from stool Support protocol for viral DNA ( , CMV) from stool Support protocol for detection of Mycobacterium tuberculosis or Legionella pneumophila in sputum or bronchoalveolar lavage Support protocol for detection of EHEC bacteria in food ( , fresh cows milk)

4 Support protocol for purification of bacterial DNA ( , Chlamydia trachomatis) from cultures, biological fluids, or clinical specimens Support protocol for purification of bacterial DNA ( , Borrelia burgdorferi) from urine Support protocol for purification of viral DNA ( , CMV) from urine Support protocol for purification of Genomic DNA from insects Support protocol for purification of Genomic DNA from dental swabs Support protocol for purification of Genomic DNA from buccal swabs 34 MACHEREY-NAGEL 01/2017, Rev.

5 174 Genomic DNA from tissue7 Appendix Troubleshooting Ordering information Product use restriction / warranty 405 MACHEREY-NAGEL 01/2017, Rev. 17 Genomic DNA from tissue1 Components Kit contentsNucleoSpin TissueREF10 Buffer T15 mL20 mL100 mLLysis Buffer B310 mL15 mL75 mLWash Buffer BW6 mL30 mL150 mLWash Buffer B5 (Concentrate)*6 mL12 mL50 mLElution Buffer BE*13 mL13 mL60 mLProteinase K (lyophilized)**6 mg30 mg2 x 75 mgProteinase Buffer mL8 mLNucleoSpin tissue Columns (light green rings)1050250 Collection Tubes (2 mL)20100500 User manual111* Composition of Elution Buffer BE: 5 mM Tris/HCl, pH ** For preparation of working solutions and storage, see section 01/2017, Rev.

6 176 Genomic DNA from Reagents, consumables, and equipment to be supplied by userReagents 96 100 % ethanolConsumables mL microcentrifuge tubes for sample lysis and DNA elution Disposable tipsEquipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Equipment for sample disruption and homogenization Personal protection equipment (lab coat, gloves, goggles) About this user manualIt is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin tissue 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.

7 All technical literature is available on the internet at Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions. Note: Buffer B3 is delivered premixed 01/2017, Rev. 17 Genomic DNA from tissue2 Product The basic principleWith the NucleoSpin tissue method Genomic DNA can be prepared from tissue , cells ( , bacteria), and many other sources. Lysis is achieved by incubation of the sample material in a proteinase K / SDS solution. Appropriate conditions for DNA binding to the silica membrane in the NucleoSpin tissue Columns are achieved by the addition of chaotropic salts and ethanol to the lysate.

8 The binding process is reversible and specific to nucleic acids. Contaminations are removed by subsequent washing with two different buffers. Pure Genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer. Kit specifications NucleoSpin tissue is designed for the rapid, small-scale preparation of highlypure Genomic DNA from any tissue , cells, bacteria, yeast, forensic samples,serum, plasma, or other body fluids. It is also suitable for preparation of DNA fromhuman or animal blood. The purified DNA can be used directly for PCR, Southernblotting, or any kind of enzymatic reactions.

9 The kit allows purification of up to 35 g of pure Genomic DNA with an A260 / A280ratio between and The NucleoSpin tissue Column is capable ofbinding up to 60 g of Genomic DNA. For lysis of certain bacterial and yeast strains, additional enzymes may benecessary which are not part of this kit. See the relevant support protocol 1: Kit specifications at a glanceParameterNucleoSpin TissueTechnologySilica-membrane technologyFormatMini spin columnSample material< 25 mg tissue 102 107 cultured cellsTypical yield20 35 gElution volume60 100 LPreparation time20 min/prep (excluding lysis)Binding capacity60 gMACHEREY-NAGEL 01/2017, Rev.

10 178 Genomic DNA from tissue Forensic quality product:NucleoSpin tissue is certified as forensic quality product. Consumables used inforensics need to be treated carefully to prevent DNA contamination. MACHEREY-NAGEL therefore has a stringently controlled production process to avoid DNAcontamination of consumables. Further, MACHEREY-NAGEL uses ethyleneoxide (EO) treatment to remove amplifiable DNA, which might still be introducedduring the manufacturing process. MACHEREY-NAGEL products carrying theforensic quality seal, contain plastic materials that are EO treated. This means,DNA of any kind, which might still be introduced into plastic consumables duringthe production process, is inactivated by means of the treatment with ethyleneoxide, in order to prevent the generation of accidental human profile by PCRamplification.