Nucleic Acid Isolation and Purification 4th edition Sea Otters Sea otters are the smallest marine mammals and are adapted to spending almost all of their lives in the ocean. Although they resemble small seals, sea otters are not actually related to seals, but rather to weasels, badgers and minks. They belong to the species „Enhydria lutris“, from the Greek „en hydra“, meaning „in the water“, and the Latin „lutris“, meaning otter. Sea otters can be found in the Pacific Ocean along the coast of California, up to Alaska, along the east coast of Russia, and all the way up to the northern tip of Japan. They prefer coastal waters and only swim out into the deep ocean when travelling between islands and the mainland. Unlike other marine mammals such as seals and dolphins, sea otters do not rely on a layer of fat to insulate their bodies from the cold water. Instead, sea otters depend on their fur to keep warm. But even with a thick fur coat, the sea otter has to work hard to keep warm, spending hours each day grooming its fur. Any water needs to be squeezed out and air needs to be blown into the fur. The air makes the fur waterproof, ensuring that the otter‘s skin remains warm and dry. That‘s why cleaning is essential. Dirty fur can easily get wet and place the otter at risk of dying from the cold. In addition to their thick coats, sea otters have another defense against the cold ocean water. The otter has a high body temperature (around +38°C), which requires a fast metabolic rate. As a result, sea otters need to eat a lot, sometimes as much as 25% of their body weight each day. This is the equivalent of humans eating about 100 hamburgers! Sea otters eat a variety of foods, including shellfish, sea urchins, fish and many different types of invertebrate sea life. As they must constantly eat to survive, otters spend a lot of time diving for food. Otters can dive up to 100 m for several minutes, although they prefer shallow waters where they can make quicker dives instead. Capturing their prey is not always an easy task. However, the sea otter is a highly intelligent animal and perfectly capable of finding ways of capturing hard-to-get prey. Smaller shellfish clinging to rocks can be prised off by the otter’s strong arms. The sea otter is fairly strong, but not quite strong enough to defeat the larger abalone, which clings fiercely to rocks. Using rocks as hammers, the otters are capable of dislodging the abalone with several firm strikes from the side. Underground food such as clams can be obtained by digging. Otters can dig with their forepaws, like dogs, and sometimes use rocks as digging implements. They have a little pouch under their left armpit in which they can store rocks or food, for example, while swimming. Once the otter has found its food, it carries it to the surface to eat. But not all food can be eaten straightaway. Although sea otters have strong teeth that can bite through crab shells and some shellfish, other sources of prey, e.g. abalones, have to be cracked open first. Individual otters seem to have their own preferences as to which method to use to crack open their prey. Some otters place a big rock on their chest and then smash their prey on the rock until the shell breaks open. Other otters reverse this operation, placing their prey on the chest and then smashing the rock against it. Some otters use numerous short, rapid strikes, while others administer a few strong blows. The sea otter’s sense of tidiness can also be observed during eating. Often the otter rolls over in the water to clear its chest of any waste scraps and avoid soiling its coat. The curious behavior of sea otters in preparing and eating their food is a perfect illustration of isolation and purification. We hope you like this little story about these cute animals. 2 Contents Chapter 1 Selection Guide ............................................................................................. 8 Introduction Product Overview ....................................................................................... 10 Overview of Nucleic Acid Purification and Isolation .................... 14 Methods described in this manual ...................................................... 16 Chapter 2 Overview of Silica Adsorption ............................................................... 18 High Pure Kits and High Pure PCR Template Preparation Kit .......................................... 21 Silica Adsorption High Pure PCR Cleanup Micro Kit ...................................................... 34 High Pure PCR Product Purification Kit ............................................. 42 High Pure Plasmid Isolation Kit ........................................................... 49 High Pure RNA Isolation Kit .................................................................. 55 High Pure RNA Tissue Kit ...................................................................... 64 High Pure FFPE RNA Micro Kit ........................................................... 69 High Pure RNA Paraffin Kit .................................................................... 75 High Pure miRNA Isolation Kit ..............................................................84 High Pure Viral RNA Kit .......................................................................... 95 High Pure Viral Nucleic Acid Kit ....................................................... 100 High Pure Viral Nucleic Acid Large Volume Kit ............................ 105 Troubleshooting Procedures for the High Pure Kits .................. 113 Agarose Gel DNA Extraction Kit ....................................................... 122 High Pure 96 UF Cleanup Kit ............................................................. 127 3 Chapter 3 Overview of Ion Exchange Chromatography ................................ 134 Ion Exchange Genopure Plasmid Midi Kit ................................................................. 136 Chromatography Genopure Plasmid Maxi Kit ................................................................ 143 Genopure Buffer Set for Low-Copy Number Plasmids ............. 150 Chapter 4 Overview of Solution-based Isolation .............................................. 152 Solution-based Isolation DNA Isolation Kit for Mammalian Blood ....................................... 154 DNA Isolation Kit for Cells and Tissues ......................................... 162 TriPure Isolation Reagent ..................................................................... 170 Chapter 5 Overview of Affinity Purification ........................................................ 180 Affinity Purification mRNA Capture Kit .................................................................................. 184 mRNA Isolation Kit ................................................................................. 189 RNA/DNA Stabilization Reagent for Blood/Bone Marrow ..... 197 mRNA Isolation Kit for Blood/Bone Marrow ................................ 198 Chapter 6 Overview of Gel Filtration ..................................................................... 208 Gel Filtration Quick Spin Columns .............................................................................. 210 mini Quick Spin Columns .................................................................... 214 Chapter 7 MagNA Lyser Instrument ...................................................................... 220 Automated Nucleic Acid MagNA Pure LC Instrument ................................................................. 222 Isolation MagNA Pure Compact Instrument .................................................... 223 4 Chapter 8 Introduction ............................................................................................... 226 Premium Performance PCR Product Selection Guide ............................................................. 227 Products for PCR and RT-PCR Product Selection Guide ...................................................... 228 RT-PCR PCR Enzyme dNTPacks ........................................................................ 229 Master Mixes ............................................................................................ 230 Real-Time PCR Instruments ............................................................... 231 Reagents for Real-Time PCR ............................................................. 232 Chapter 9 Tips for Handling Nucleic Acids ........................................................ 234 Appendix Conversion Tables and Formulas ...................................................... 236 Buffers and Gels for Electrophoresis ............................................... 240 Other Useful Information ..................................................................... 242 Chapter 10 Isolation and Purification of DNA ..................................................... 246 Ordering Guide Isolation and Purification of RNA ..................................................... 247 MagNA Lyser Instrument and Accessories.................................... 248 Automated Isolation using the MagNA Pure LC Instrument ....248 Automated Isolation using the MagNA Pure Compact Instrument ....................................................................................................249 Companion Reagents for Isolating Nucleic Acids ........................250 Chapter 11 ......................................................................................................................... 255 Index 5 Disclaimer * For life science research only. Not for use in diagnostic procedures. + For general laboratory use. # CE/for USA for laboratory use 6 1 Introduction Selection Guide 8 Product Overview 10 Overview of Nucleic Acid Purification and Isolation 14 Methods described in this manual 16 Selection Guide Selection Guide Use this table to select a product according to the type of nucleic acid you wish to purify, then consider the 1 source of the nucleic acid and the scale of the purification. Nucleic Subtype Origin/Source Scale Recommended Product see Acid Page Type tissue, cultured cells, bacteria, yeast, blood,  High Pure PCR Template Preparation Kit 21 dried blood spots tissue, cultured cells, bacteria, yeast, mouse   DNA Isolation Kit for Cells and Tissues 162 Genomic tail human blood, dried blood spots  High Pure PCR Template Preparation Kit 21 mammalian/human blood   DNA Isolation Kit for Mammalian Blood 154 propagated in E. coli  High Pure Plasmid Isolation Kit 49 Plasmid propagated in E. coli  Genopure Plasmid Midi Kit 136 propagated in E. coli  Genopure Plasmid Maxi Kit 143 serum, plasma, blood, other body fluids,  High Pure Viral Nucleic Acid Kit 100 supernatant from cell cultures Viral serum, plasma, whole blood   High Pure Viral Nucleic Acid Large 105 Volume Kit DNA PCR mixture  High Pure PCR Product Purification Kit 42 • High Pure PCR Cleanup Micro Kit 34  High Pure 96 UF Cleanup Kit 127 restriction enzyme digests, labeling and  High Pure PCR Product Purification Kit 42 modifying reaction mixture • High Pure PCR Cleanup Micro Kit 34 DNA radiolabeled DNA  Quick Spin Columns 210 fragments radiolabeled DNA, removal of excess  mini Quick Spin Columns 214 fluorescent-labeled terminators agarose gel slices   Agarose Gel DNA Extraction Kit 122  High Pure PCR Product Purification Kit 42 • High Pure PCR Cleanup Micro Kit 34 ss DNA, ds DNA, PCR products, cRNA • High Pure PCR Cleanup Micro Kit 34   Scale: •  micro mini midi maxi 8 Nucleic Acid Isolation and Purification Manual Selection Guide Selection Guide, continued 1 Nucleic Subtype Origin/Source Scale Recommended Product see Acid Page Type cultured cells, tissues, total RNA   mRNA Isolation Kit 189 •  mRNA Capture Kit 184 mRNA whole blood/bone marrow    mRNA Isolation Kit for Blood/Bone 198 Marrow cultured cells, bacteria, yeast, blood  High Pure RNA Isolation Kit 55 tissue  High Pure RNA Tissue Kit 64 cultured cells, tissues, bacteria, yeast, blood,    TriPure Isolation Reagent 170 Total plant cells RNA RNA formalin-fixed, paraffin-embedded tissue • High Pure FFPE RNA Micro Kit 69 formalin-fixed, paraffin-embedded tissue,  High Pure RNA Paraffin Kit 75 fresh-frozen tissue animal tissue, stabilized animal tissue,  High Pure miRNA Isolation Kit 84 animal cell culture, formalin-fixed, paraffin-embedded tissue serum, plasma, other body fluids, supernatant  High Pure Viral RNA Kit 95 Viral RNA from cell cultures radiolabeled RNA  Quick Spin Columns 210 RNA fragments  mini Quick Spin Columns 214   Scale: •  micro mini midi maxi Introduction 9 Product Overview Product Overview Detailed Product Characteristics Recommended Uses Product Overview Detailed Product Characteristics Purification method/Product Starting material and quantity Yield/recovery Time required PCR/ RT-PCR DD-RT- cDNA RE Southern Labeling Northern RNase CloningSequen- in vitro in Trans- Micro - 1 long PCR Synthesis/ digestion blotting modifying blotting protection cing trans- vitro fection array 1 PCR primer reactions assays cription trans- Spotting extension lation Ultrafiltration High Pure 96 UF Cleanup Kit PCR products (100 bp to > 10 kb), 25 µl (150 bp, 40%; 1500 bp, < 20 min      20-300 µl 90%; 4500 bp, 90%, 8000 bp, 80%) Silica Adsorption High Pure PCR Template Preparation Kit* blood, 200 – 300 µl 3 – 9 µg 20 min     cultured cells, 104 – 108 15 – 20 µg 20 min thymus tissue, 25 – 50 mg 5 – 20 µg 80 min, incl. lysis mouse tail, 25 – 50 mg 5 – 10 µg 200 min, incl. lysis yeast, 108 cells 10 – 13 µg 50 min, incl. lyticase digest bacteria, 109 cells 1 – 3 µg 35 min, incl. lysozyme digest dried blood spots 9 mm punch-out 80 min, incl. lysis High Pure PCR Product Purification Kit PCR, modifying, labeling, restriction >80% recovery of 5 – 25 µg DNA 10 min       digestion reaction, 100 µl; agarose gel >100 bp slices High Pure PCR Cleanup Micro Kit PCR, modifying, labeling, restriction >80% recovery of 5 – 25 µg DNA 10 min       digestion reaction, agarose gel slices High Pure Plasmid Isolation Kit* E. coli XL1 blue, pUC 19 (2 ml) 12 µg 30 min        E. coli HB 101, pUC 19 (2 ml) 6 µg 30 min E. coli DH5, pUC 19 (2 ml) 3.5 µg 30 min High Pure RNA Isolation Kit* blood, 200 – 500 µl sufficient for 10 RT-PCR reactions 50 min, incl. RBC lysis       cultured cells, 106 20 µg 25 min yeast, 108 cells 20 µg 45 min, incl. lyticase digest bacteria, 109 cells 35 – 50 µg 90 min, incl. lysozyme digest High Pure RNA Tissue Kit solid tissue, 1 – 10 mg 0.5 – 3.0 µg/mg 30 min       High Pure miRNA Isolation Kit tissue, stabilized tissue, cell culture, depending on miRNA 30 min    FFPE tissue sections High Pure Viral RNA Kit serum, plasma, urine, supernatant from product detectable by RT-PCR 10 min  cell culture, 200 – 600 µl High Pure Viral Nucleic Acid Kit* serum, plasma, blood, supernatant from product detectable by PCR or 20 min   cell culture, RT-PCR 200 – 600 µl High Pure 16 System Viral Nucleic Acid serum, plasma, supernatant from cell product detectable by PCR or 30 min   Kit culture, 200 µl RT-PCR High Pure Viral Nucleic Acid Large serum, plasma, whole blood 1 – 2.5 ml 25 min   Volume Kit High Pure RNA Paraffin Kit fresh-frozen or formalin-fixed paraffin- 0.3-1.5 µg/5 µm section, 2 h without overnight    embedded tissue 2-6 µg/20 mg incubation fresh-frozen tissue High Pure FFPE RNA Micro Kit 1-10 µm FFPE tissue sections 1.5-3.5 µg/5 µm 60 min without 3h    incubation Agarose Gel DNA Extraction Kit agarose gel slices, 100-200 mg 80% recovery of fragments 60 min      (0.4-9.5 kb) Ion Exchange Chromatography Genopure Plasmid Midi Kit E.coli DH5, pBS (30 ml) >85 µg 60 min       Genopure Plasmid Maxi Kit E.coli DH5, pBS (150 ml) >420 µg 75 min       Affinity Purification mRNA Isolation Kit for Blood/ blood, bone marrow aspirate, 50 – 200 ng/ml blood 90 min      Bone Marrow* 1.5 – 5 ml mRNA Isolation Kit* tissue, 50 mg – 1 g 7 – 14 µg/100 mg tissue 60 min      cells, 2 x 105 – 108 0.3 – 25 µg/107 cells 40 min total RNA, 250 µg – 2.5 mg 1 – 5 µg/100 µg total RNA 30 min mRNA Capture Kit tissue, up to 20 mg product detectable by RT-PCR 60 min  cells, up to 5 x 105 product detectable by RT-PCR 40 min total RNA, up to 40 µg product detectable by RT-PCR 30 min 10 Nucleic Acid Isolation and Purification Manual Introduction 11