SurePrep Purification Kits for Isolating RNA, DNA and Proteins. Clinical, Forensics, and Medical Research

Transcription

1 SurePrep Purification Kits for Isolating, DNA and Proteins Clinical, Forensics, and Medical Research

2 CONTENTS SurePrep Application Summary Chart...2 SurePrep FFPE Isolation Kit...3 SurePrep Nuclear or Cytoplasmic Purification Kit...4 SurePrep /DNA/Protein Purification Kit...5 SurePrep Small Purification Kit...6 SurePrep TrueTotal Purification Kit...7 SurePrep /Protein Purification Kit...8 SurePrep Leukocyte Purification Kit...9 SurePrep Cleanup and Concentration Kit SurePrep Urine Exfoliated Cell Purification Kit SurePrep Urine Bacterial Purification Kit BP BP BP BP BP BP BP BP BP BP FFPE Cytoplasmic, DNA, and Protein Small TrueTotal and Protein Leukocyte Cleanup and Concentration Urine Exfoliated Cell X X X X X X X X X micro X X X X X X X X X X DNA Protein X X X Urine Bacterial Laboratory Applications Agricultural X X X X X Environmental X X X X X Food Science X X X X X Forensic Pathology X X X X X X and Biology Histology X Industrial and Public Health X X X X X X X X X X Infectious Disease Detection X X X X X X X X X X Industries Medical Research Academic Research Government Research Biotech Research X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

3 SurePrep FFPE ISOLATION KIT APPLICATION SurePrep FFPE Isolation Kits rapidly isolate and purify total from formaldehyde-fixed paraffinembedded (FFPE) tissue samples. Using formalin to fix tissues results in crosslinking of and protein. Moreover, the process of embedding the tissue samples can also lead to fragmentation of the over time. SurePrep FFPE Isolation Kits provide conditions to partially reverse the formalin modifications, maximizing the yield of functional. Higher yield of contributes to greater accuracy of results in downstream applications. SurePrep FFPE Isolation Kits are well suited for forensic pathology and biology, histology, medical research and academic research. Rapid method for the isolation and purification of all sizes of, ranging from large m and ribosomal down to micro (mi) and small interfering (si) less than 200 nt* An easy-to-follow, streamlined protocol of five simple steps allows for efficient purification without the use of phenol and chloroform An innovative procedure to overcome the formalin modifications resulting in high yields of functional Spin column format for fast and easy processing of samples. Optional protocol for on-column DNA removal High yields of purified total for use in downstream applications such as qrt-pcr qrt-pcr Primer extension Gene expression array assays FFPE Tissue Samples Deparaffinization with xylene. Wash with ethanol Add digestion buffer, proteinase K. Incubate Add Binding Solution, Ethanol Bind Wash Elute Procedure for BP , SurePrep FFPE Isolation Kit Four-hour procedure Purified Total SurePrep FFPE ISOLATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg Maximum Amount of Sample... 5 paraffin slices, 20µm thick Unsectioned block: 25mg Size of Purified... Large, small and micro. Protocol does not assume fragmentation. Lysis Method... Deparaffinization with xylene followed by enzymatic digestion Final Elution Volume... 50µL No. of Buffers... 5 buffers, 2 enzymes Time to Complete Process... 4 hours (3-hour proteinase K digestion) 50 BP SurePrep FFPE Isolation Purification Kit Note: Longer time is required to sufficiently isolate all functionally intact total species from a preserved sample in order to maximize yield. * The fragments purified depend on the age of the FFPE tissue as the degree of fragmentation of the will increase over time. 3

4 SurePrep NUCLEAR OR CYTOPLASMIC PURIFICATION KIT SurePrep Nuclear or Cytoplasmic Purification Kits rapidly isolate and purify both nuclear and cytoplasmic from cultured animal cells and small tissue samples. SurePrep kits are well suited for academic and medical research. Fast and easy processing using efficient spin-column procedure All nuclear and cytoplasmic molecules can be isolated, from large m to micro (mi) and small interfering (si) Isolate fractionated as opposed to total No genomic DNA contamination in the cytoplasmic fraction Total is isolated without the use of phenol and chloroform Real-time PCR Reverse transcription PCR RNase protection and primer extension Expression array analysis Supernatant (Cytoplasmic Fraction) Lyse Cells (Monolayer, cell suspension, lifted cells or tissues) Add Binding Solution Add Ethanol Centrifuge to separate cell fractions Centrifuge to clear lysate 1. Bind 2. Wash 3. Elute Pellet (Nuclear Fraction) Add Binding Solution Add Ethanol Centrifuge to clear lysate 1. Bind 2. Wash 3. Elute Procedure for BP , SurePrep Nuclear or Cytoplasmic Purification Kit Forty-five minutes for 10 purifications Cytoplasmic Nuclear SurePrep NUCLEAR OR CYTOPLASMIC PURIFICATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg Size of Purified... All sizes, including small (<200 nt) Maximum Amount of Starting Material Animal Cells... 3 x 10 6 cells Animal Tissues... 15mg Time to Complete Purification Process minutes for 10 purifications Average Yields from Purification HeLa Cells, Cytoplasmic (1 x 10 6 cells)... 15µg HeLa Cells, Nuclear (1 x 10 6 cells) µg Lysis Solution, 20mL; Wash Solution, 22mL; Binding Solution, 25mL; Elution Buffer, 6mL; Spin Columns, 50; Collection Tubes, 50; 1.7mL Elution Tubes, 50; Product Insert All solutions should be kept tightly sealed and stored at room temperature BP SurePrep Nuclear or Cytoplasmic Purification Kit

5 SurePrep /DNA/PROTEIN PURIFICATION KIT APPLICATION SurePrep /DNA/Protein Purification Kits rapidly isolate and purify total, genomic DNA and total proteins sequentially from a single sample. Samples may consist of cultured animal cells, tissue samples, blood, bacteria, yeast, fungi or plants. These kits are useful for isolating macromolecules from difficult-to-obtain or small samples such as biopsy materials because the SurePrep process eliminates the need to fractionate the sample. SurePrep kits are well suited for academic and medical research. Fast and easy processing using efficient spin-column procedure Total, genomic DNA and proteins are all column-purified using the same column Sequential isolation of nucleic acids and proteins from a single lysate: no need to split the lysate Isolate high-quality in all sizes, including large m and r to micro (mi) Analysis is more reliable since, DNA and proteins are derived from the same sample Isolate high-quality genomic DNA with a molecular weight 30kb High yields of isolated proteins No phenol or chloroform extractions qrt-pcr RNase protection Primer extension Expression array analysis GENOMIC DNA PCR Sequencing Southern blotting SNP analysis PROTEIN SDS-PAGE analysis Western blots Mass Spectrometry A. Purification of and DNA Lyse cells or tissue using Lysis Solution Bind and DNA to Column Wash Elute Wash DNA Elute gdna gdna Flowthrough (Proteins) Adjust ph B. Purification of Proteins Proteins Bind Proteins to Activated Column Wash Elute Proteins Procedure for BP , SurePrep /DNA/Protein Purification Kit Thirty minutes for 10 purifications SurePrep /DNA/PROTEIN PURIFICATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg for 20µg for DNA 200µg for protein Size of Purified... All sizes, including small (<200 nt) Size of DNA Purified... 30kb Maximum Amount of Starting Material Animal cells... 3 x 10 6 cells Animal tissues... 25mg Blood µL Bacteria... 1 x 10 9 cells Yeast... 1 x 10 8 cells Fungi... 50mg Plant Tissues... 50mg Time to complete purification process...30 minutes for 10 purifications Average Yields from Purification HeLa Cells (1 x 10 6 cells)... 15µg HeLa Cells (1 x 10 6 cells)... 8µg DNA HeLa Cells (1 x 10 6 cells) µg protein Lysis Solution, 40mL; Wash Solution, 20mL; Elution Solution, 6mL; gdna Wash Solution, 15mL; gdna Elution Buffer, 15mL; Protein Column Regeneration Buffer, 30mL; Protein Column Activation and Wash Buffer, 60mL; Protein ph Binding Buffer, 4mL; Protein Elution Buffer, 8mL; Protein Neutralizer, 2mL; Protein Loading Dye, 2mL; Spin Columns, 50; Collection tubes, 200; Product Insert The Protein Loading Dye should be stored at -20 C upon arrival. All other solutions should be kept tightly sealed and stored at room temperature. 50 BP SurePrep / DNA/Protein Purification Kit

6 SurePrep SMALL PURIFICATION KIT SurePrep Small Purification Kits are optimized for isolation and purification of small s (< 200 nt) from larger s in various biological samples such as animal cells and tissues, bacteria, and plants. These kits are well suited for academic and medical research. Isolate all small molecules (<200 nucleotides) Minimal contamination from large molecules and genomic DNA Fast and easy processing using rapid spin-column format No phenol or chloroform extractions Quantitative RT-PCR Microarray assays 200 nt Lyse cells or tissue using Lysis Solution Centrifuge to clear lysate (Optional) Add Ethanol Bind large molecules to Large Removal Column Retain Flowthrough Add Ethanol to flowthrough Bind small molecules from flowthrough onto Small Enrichment Column Wash three times with Wash Solution Procedure for BP , SurePrep Small Purification Kit Forty-five minutes for 10 purifications Small isolated from 5 x 10 5 HeLa cells using the SurePrep Small Purification Kit. Purified small (lane 1) and a 100 nt ladder (lane 2) were run on an 8% urea-page gel. 100 nt Elute small with Elution Buffer Purified Small (mi, si, t, 8S r, etc.) SurePrep SMALL PURIFICATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg Size of Purified... <200 nt Maximum Amount of Starting Material Animal Cells... 3 x 10 6 cells Animal Tissues... 25mg Bacteria... 5 x 10 8 cells Plant Tissues... 50mg Time to Complete Purification Process minutes for 10 purifications Lysis Solution, 2 x 20mL; Wash Solution, 22mL; Elution Buffer, 6mL; Large Removal Columns, 25; Small Enrichment Columns, 25; Collection Tubes, 50; 1.7mL Elution Tubes, 50; Product Insert Store at room temperature BP SurePrep Small Purification Kit

7 SurePrep TrueTotal PURIFICATION KIT SurePrep TrueTotal Purification Kits rapidly isolate and purify total from a variety of biological sources including blood, animal cells and tissues, bacteria, yeast, fungi, and plants. SurePrep kits are well suited for academic and medical research. Fast and easy processing using rapid spin-column format Isolate all sizes of including large and small species No phenol or chloroform extractions Isolate and detect from as little as a single animal cell Quantitative RT-PCR RNase protection and primer extension Expression array assays Total isolated from E. coli in log-phase growth using the TrueTotal Purification Kit. One µg of total isolated from E. coli (strain FB5!, Fisher Cat. # BP4000-3) was loaded in lanes 2-4 on a 1.5% denaturing formaldehyde-agarose gel. Lane 1 contained a 1kb standard (BP2811). Electrophoresis was performed at 100V for 70 min. and the gel stained with ethidium bromide. 200 nt 23S r 16S r Diverse species of Small < 200 nt Centrifuge to clear lysate Lyse cells or tissue Purified Total Add Ethanol 1. Bind to Column 2. Wash Column 3. Elute Procedure for BP , SurePrep TrueTotal TM Purification Kit Twenty minutes for 10 purifications SurePrep TrueTotal PURIFICATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg Size of Purified... All sizes, including small (<200 nt) Maximum Amount of Starting Material Animal Cells... 3 x 10 6 cells Animal Tissues... 25mg Blood µL Bacteria... 1 x 10 9 cells Yeast... 1 x 10 8 cells Fungi... 50mg Plant Tissues... 50mg Time to Complete Purification Process minutes for 10 purifications Average Yields from Purification HeLa cells (1 x 10 6 cells)... 15µg E. coli (1 x 10 9 cells)... 50µg Lysis Solution, 40mL; Wash Solution, 22mL; Elution Buffer, 6mL; Micro Spin Columns, 50; Collection Tubes, 50; 1.7mL Elution Tubes, 50; Product Insert Store at room temperature. 50 BP SurePrep TrueTotal Purification Kit 7

8 SurePrep /PROTEIN PURIFICATION KIT SurePrep /Protein Purification Kits rapidly isolate and purify total and proteins sequentially from a single sample of cultured animal cells, tissue samples, blood, bacteria, yeast, fungi or plants. SurePrep kits are well suited for academic and medical research. Fast and easy processing using rapid spin-column format All columns for purification and protein purification provided Sequentially isolate nucleic acids and proteins from a single lysate: no need to split the lysate Isolate all sizes of, from large m and r down to micro (mi) No phenol or chloroform extractions Isolate high quality total High yields of isolated proteins PROTEIN Reverse transcription PCR SDS-PAGE analysis Western blots RNase protection Primer extension Expression array analysis A. Purification of Lyse cells or tissue Bind to Column Wash Elute Flowthrough (Proteins) Adjust ph B. Purification of Proteins Proteins Bind Proteins to Activated Column Wash Elute Proteins Procedure for BP , SurePrep /Protein Purification Kit Thirty minutes for 10 purifications SurePrep /PROTEIN PURIFICATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg 200µg for protein Size of Purified... All sizes, including small (<200 nt) Maximum Amount of Starting Material Animal Cells... 3 x 10 6 cells Animal Tissues... 25mg Blood µL Bacteria... 1 x 10 9 cells Yeast... 1 x 10 8 cells Fungi... 50mg Plant Tissues... 50mg Time to complete purification process minutes for 10 purifications Average Yields from Purification HeLa Cells (1 x 10 6 cells)... 15µg HeLa Cells (1 x 10 6 cells) µg protein Lysis Solution, 40mL; Nucleic Acid Wash Solution, 22mL; Nucleic Acid Elution Solution, 15mL; Protein Column Regeneration Buffer, 30mL; Protein Column Activation and Wash Buffer, 60mL; Protein ph Binding Buffer, 4mL; Protein Elution Buffer, 8mL; Enzyme Incubation Buffer, 6mL; Protein Neutralizer, 2mL; Protein Loading Dye, 2mL; Spin Columns, 50; Collection tubes, 150; Product Insert The Protein Loading Dye should be stored at -20 C upon arrival. All other solutions should be kept tightly sealed and stored at room temperature BP SurePrep /Protein Purification Kit

9 SurePrep LEUKOCYTE PURIFICATION KIT SurePrep Leukocyte Purification Kits rapidly isolate and purify total leukocyte (white blood cell) from mammalian blood samples. The purified can be used in various expression studies including those focusing on diseases. SurePrep kits are well suited to academic and medical research. Fast and easy processing using rapid spin-column format Differential red blood cell lysis allows for removal of a majority of globin ms Isolate total leukocyte, including all small species No phenol or chloroform extractions High-quality leukocyte can be used in a number of downstream applications Reverse transcription PCR RNase protection and primer extension Expression array analysis Procedure for Differential Red Blood Cell (RBC) Lysis Collect Blood in 4.8mM EDTA Add 5 Volumes of RBC Lysis Buffer. Vortex and incubate for 3-5 minutes. 1. Centrifuge to pellet cells 2. Gently decant supernatant Add 2 Volumes of RBC Lysis Buffer. Vortex 1. Centrifuge to pellet cells 2. Gently decant supernatant White Leukocyte Pellet Procedure for Total Leukocyte Purification Lyse leukocyte pellet using Binding Solution Add Ethanol Centrifuge to clear lysate Bind Wash Elute Procedure for BP , SurePrep Leukocyte Purification Kit Forty-five minutes for 10 purifications White Leukocyte Pellet Total Leukocyte SurePrep LEUKOCYTE PURIFICATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg Size of Purified... All sizes, including small (<200 nt) Maximum Amount of Starting Material Maximum Blood Input... 2mL or 3 x 10 6 Leukocytes Minimum Blood Input... 10µL Time to Complete Purification Process minutes for 10 purifications Average Yields from Purification 500µL human blood µg RBC Lysis Solution, 180mL; Binding Solution, 25mL; Wash Solution, 22mL; Elution Buffer, 6mL; Spin Columns, 50; Collection Tubes, 50; 1.7mL Elution Tubes, 50; Product Insert All solutions should be kept tightly sealed and stored at room temperature. The RBC Lysis Solution should be stored at 4 C upon arrival. 50 BP SurePrep Leukocyte Purification Kit 9

10 SurePrep CLEANUP AND CONCENTRATION KIT SurePrep Cleanup and Concentration Kits rapidly purify and concentrate up to 50µg of isolated from other methods including phenol/guanidine-based protocols, and from various upstream enzymatic reactions such as DNase treatment, labeling, and in vitro transcription. SurePrep kits are well suited to academic and medical research. Fast and easy processing using rapid spin-column format Suitable for all sizes of, from large m to micro- (mi) Able to clean up isolated using different methods, including from phenol/chloroform extractions qrt-pcr RNase protection and primer extension Expression array analysis Vortex to mix Sample Purified Total Add Binding Solution and Ethanol Bind to column Wash Elute Procedure for BP , SurePrep Cleanup and Concentration Kit Twenty minutes for 10 purifications SurePrep CLEANUP AND CONCENTRATION KIT - SPECIFICATIONS Column Binding Capacity... 50µg Size of Purified... All sizes, including small (<200 nt) Maximum Amount of Starting Material... 50µg of Minimum Elution Volume... 20µL Time to Complete Purification Process minutes for 10 purifications Average Recovery... 90% Binding Solution, 2 x 20mL; Wash Solution, 22mL; Elution Buffer, 6mL; Spin Columns, 50; Collection Tubes, 50; 1.7mL Elution Tubes, 50; Product Insert All solutions should be kept tightly sealed and stored at room temperature BP SurePrep Cleanup and Concentration Kit

11 SurePrep URINE EXFOLIATED CELL PURIFICATION KIT SurePrep Urine Exfoliated Cell Purification Kits rapidly isolate and purify total from exfoliated cells shed into the urine from the urinary tract. Purified biomarkers from exfoliated cells can be used as non-invasive tools for a number of diagnostic and research applications such as monitoring bladder, kidney, or other urinary tract disorders. SurePrep kits are well suited for academic and medical research. Fast and easy processing using efficient spin-column procedure Isolate and detect all sizes of total, from large m and ribosomal down to micro (mi) using as little as 1mL sample of urine can be isolated and detected from as few as 100 exfoliated cells Isolate high-quality total from urine Total is isolated without the use of phenol and chloroform SurePrep URINE BACTERIAL PURIFICATION KIT SurePrep Urine Bacterial Purification Kits isolate from both Gram negative and Gram positive bacteria, including E. coli, Proteus spp., Klebsiella spp., Enterobacter spp., Serratia spp., Pseudomonas spp., Clostridial ssp., and Leptospirosis spp., as well as Chlamydia trachomatis and Neisseria gonorrhoeae from either human or animal urine samples. These kits preferentially purify from cellular proteinaceous components, as well as glucose and salts found in urine. SurePrep kits are well suited to academic and medical research. Fast and easy processing of multiple samples in 30 minutes using efficient spin-column procedure Isolate a diversity of high-quality total, from large m and ribosomal down to micro and small interfering Total is isolated without the use of phenol and chloroform Centrifuge to clear lysate (Optional) Urine Sample Pellet bacterial cells Lyse cells using TE buffer with lysozyme and lysis solution. Add Ethanol Bind to column Wash Elute Purified Total Urine Bacterial Procedure for BP , SurePrep Urine Bacterial Purification Kit Thirty-minute procedure. SurePrep URINE EXFOLIATED CELL and SurePrep URINE BACTERIAL PURIFICATION KITS - SPECIFICATIONS Column Binding Capacity... 50μg Volume of Urine Processed mL, Exfoliated Cell Kit (BP ) 10-50mL, Bacterial Kit (BP ) Maximum Input of Exfoliated Cells x 10 6 cells Exfoliated Cell Kit (BP ) Maximum Column Loading Volume μL Size of Purified... All sizes, including small (<200 nt) Time to Complete 10 Purifications min. for Exfoliated Cell Kit (BP ) 30 min. for Bacterial Kit (BP ) Average Yields from Purification*... ~1μg per 1 x 10 5 cells, Exfoliated Cell Kit (BP ) ~0.5μg per 1 x 10 7 cells, Bacteria Kit (BP ) *Yield varies due to cell density of sample. 50 BP SurePrep Urine Exfoliated Cell Purification Kit Reverse Transcription PCR RNase protection and primer extension Expression array assays Lysis Solution, 40mL; Wash Solution, 22mL; Elution Solution, 6mL; Spin Columns, 50; Collection Tubes, 50; 1.7mL Elution Tubes, 50; Product Insert All solutions should be kept tightly sealed and stored at room temperature. 50 BP SurePrep Urine Bacterial Purification Kit 11

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