Photo: istockphoto.com Vizerskaya. Reaction No.1

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1 Photo: istockphoto.com Vizerskaya Reaction No.1 Selected Life Science Products 2014

2 2 ReAction In the center of molecular biology is one species of molecules: DNA. DNA molecules are amplified and introduced into organisms by transformation or transfection, separated, stained, examined under the microscope, manipulated, sequenced and so on. For all these techniques the initial step is to isolate DNA from the origin of interest. Nucleic Acid Isolation CTAB Lysis buffer BioChemica Cetyltrimethylammonium bromide (CTAB) is used to liberate and form complexes with total nucleic acids of plants or fungi; polysaccharides, phenolic compounds and other enzyme-inhibiting contaminats found in A4150, L plant cells are efficiently removed in the supernatant. Lysozyme BioChemica A3711, g Proteinase K* A3830, mg Proteinase K - Solution A4392, ml Cesium chloride % A1098, g 1-Bromo-3-chloropropane BioChemica Chloroform substitute in phenolic DNA extraction. A2107, ml Bromo chloropropane is less toxic than chloroform and forms a tighter interphase! Chloroform BioChemica A3691, ml Isoamyl alcohol A2610, ml TRItidy G Ready-to-use monophasic reagent (contains phenol and guanidinium thiocyanate) for subsequent A4051, ml isolation of RNA, DNA and proteins from samples of human, animal, plant and bacterial origin. RNase A (Dnase-free) A3832, mg Phenol equilibrated, stabilized A1153, ml Phenol equilibrated, stabilized: Chloroform:Isoamyl alcohol 25 : 24 : 1 For DNA isolation A0889, ml DNA Isolation reagent for genomic DNA Non-organic and ready-to-use reagent for the isolation of genomic DNA from human, animal (incl. mouse tail), plant, yeast, bacterial and viral origin. The isolated DNA can be used, without additional purification, for A3418, ml Southern-analysis, dot blot hybridization, molecular cloning, RFLP, PCR and other molecular biology and biotechnology applications. DEPC BioChemica A0881, ml DNase I A3778, mg A3778, mg Phenol water-saturated, stabilized A1624, ml Phenol stabilized: Chloroform:Isoamyl alcohol 25 : 24 : 1 A2279, ml RNAtidy G Ready-to-use solution for the isolation of small and large RNA species ( kb) from biological material with high purity (DNA and protein-free). Mono-phasic reagent containing phenol and guanidinium thiocyanate. A2867, ml RNase-ExitusPlus A7153, ml RNase decontamination solution; removes RNase contaminations from surfaces by non-enzymatic degradation. A7153, ml * Proteinase K When it comes to protein degradation, proteinase K is the most common protease directly employed in the lysis solution. Conditions that promote enzyme inactivation like detergents, chaotropic salts, high temperature and changes in ph are well tolerated by proteinase K. Beside its high stability, proteinase K is characterized by a large number of cleavage sites and therefore perfectly suited to remove cellular and nuclear proteins that are attached to the DNA. Furthermore, the permissive Proteinase K has no need for cofactors, so it can t be inhibited by EDTA.

3 AppliChem s Solution! Nucleic Acid Decontamination with DNA-ExitusPlus DNA contamination of PCR workstations leads to faulty DNA amplifications. Hidden traces of DNA may cause false diagnoses or contamination of sequence databases. Control and elimination of unwanted DNA background therefore is mandatory for quality control in all PCR laboratories. Our patented product DNA-ExitusPlus employs a mild and non-corrosive chemistry for rapid nonenzymatic degradation of nucleic acids. Even a short incubation time with DNA-ExitusPlus completely removes non-target DNA and RNA from work surfaces and tools (1, 2). There are two different versions of DNA-Exitus Plus available: DNA-ExitusPlus (A7089) includes a color indicator to easily visualize the surface area covered by the reagent. DNA-ExitusPlus IF (A7409) is almost colorless. DNA-ExitusPlus is a registered trademark of AppliChem GmbH. Products for Nucleic Acid Decontamination DNA-ExitusPlus A7089, ml DNA-ExitusPlus A7089, ml DNA-ExitusPlus IF A7409, ml DNA-ExitusPlus IF A7409, ml Autoclave-ExitusPlus A7600,1000 Powder for 6 x 1 L DNA-free Reagents for PCR Water tested PCR, DNA-free SYBR Green -staining solution, DNA-free A8510, x 1,7 ml A8511, x 0,625 ml

4 4 ReAction Verify the effectiveness of decontamination agents It s harder than it seems to test decontamination solutions for a complete DNA degrading activity. It is not sufficient at all to just mix decontamination solution and DNA and to perform a subsequent PCR to amplify residual DNA. Since PCR is too easily affected by factors such as unfavorable buffer conditions, PCR inhibitors or DNA cross contamination. But for the evaluation of the potential of a DNA decontamination reagent, one has to use PCR analysis in combination with a sensitive DNA degradation test. We have developed a DNA strand break assay which allows us to evaluate the efficiency of DNA-ExitusPlus [1]. A. The DNA strand break assay in brief: 1. Incubate for 3-10 min 200 ng of plasmid DNA with water (C), commercial decontamination solutions (X1-X4) and DNA-ExitusPlus (D). 2. Denature all samples with heat. 3. agarose gel electrophoresis of DNA and degradation products. B. The PCR test in brief: 1. Incubate 0.1 to 1 ng of test DNA with water (A) or DNA-ExitusPlus (B). 2. Wash twice the reactions. 3. Add a control DNA, and 4. Run PCR with primers against test and control DNA. 5. Agarose gel electrophoresis of PCR products. Results While the control treatment and all tested competitors show no DNA degradation, plasmid DNA is degraded completely after 10 min of treatment with DNA-ExitusPlus. 3 min of DNA-ExitusPlus -treatment leads already to a strong fragmentation of the test plasmids. Results After incubation with DNA-ExitusPlus no PCR amplicons for the test DNA can be detected indicating all test DNA template has been degraded. The PCR product for control DNA confirms functionality of the PCR. Literature [1] Esser et al. (2006) DNA Decontamination: Novel DNA-ExitusPlus in comparison with conventional reagents. BioTechniques 40 (2), On closer inspection, only DNA-ExitusPlus destroys DNA completely. [2] Arena, A. (2010) Dna Exitus Plus Versus Standard Bleach Solution for the Removal of Dna Contaminants on Work Surfaces and Tools. Investigative Science Journal 2, DNA-ExitusPlus proved as effective as freshly prepared bleach solution in an assay using swap tests and subsequent qpcr. Please see our website or refer to our AppliCation No.1 for more details of the procedure.

5 Agarose gel electrophoresis ReAction 5 DNA ladder 1 kb (A3470) The mass of every band is defined and may be used for semi-quantitative determination of DNA concentration. Decontamination Bags (A9676) in use Just add a tea bag to the ethidium bromide-containing buffer and wait... activated charcoal will absorb the dye. Ethidium bromide dropper-bottle A2273 One drop of the solution stains an agarose gel of 50 ml. The dropper-bottle is convenient and minimizes the possible contact with ethidium bromide. DNA-Dye NonTox Non-hazardous, non-mutagenic fluorescent dye (ethidium bromide substitute) for staining of DNA in A9555, ml agarose gels. Detection under blue light or UV light. Supplied as a ready-to-use 6X loading dye. Ethidium bromide - Solution 1 % BioChemica A1152, ml Ethidium bromide - Solution 0.07 % dropper-bottle A2273, ml Decontamination Bag Tear-resistant charcoal bags for removal of DNA dyes such as ethidium bromide, A9676, Stk. SYBR Green or propidium iodide from aqueous solutions. Loading buffer DNA I Contains bromophenol blue and Ficoll 400 A3144, ml Loading buffer DNA II Contains bromophenol blue, xylene cyanol FF and Ficoll 400 A2571, ml Loading buffer DNA IV (for Agarose gels) Contains bromocresol green, xylene cyanol, EDTA, SDS and Ficoll 400 A3481, ml Agarose Basic A8963, kg Agarose high EEO A2115, g Agarose low EEO (Agarose Standard) A2114, g Agarose Low Melt Large DNA grade For analytic and preparative DNA gels A3762, g Agarose medium EEO A2116, g Agarose MP Multi-purpose agarose; very good analytic separation (100 bp to 50 kb!); blotting; DNA typing; PFGE. A1091, g DNA Ladder 1 kb A5207, µg DNA Ladder 100 bp A5191, µg DNA Ladder 100 bp (lyophilised) A3470, µg DNA Ladder 50 bp A8368, µg DNA Ladder Mix (lyophilised) A3660, µg

6 6 ReAction Biochemistry Enzyme substrates 2-Nitrophenyl-ß-D-galactopyranoside BioChemica A1272, g 4-Nitrophenyl phosphate disodium salt hexahydrate BioChemica A1442, g NADPH tetrasodium salt A1395, g NADP sodium salt A1394, g NAD A1124, g L-Glutathione oxidize BioChemica A2243, g L-Glutathione reduced BioChemica A2084, g ß-Glycerol phosphate disodium salt pentahydrate BioChemica A2253, g X-Gal Molecular Biology grade A4978, g IPTG Molecular Biology grade A4773, g example Denaturating, reducing, complexing Guanidine thiocyanate-solution (6 M in 0.1 M Tris; ph 7.5) A0703, L Guanidine thiocyanate A1107, g Guanidine hydrochloride A1106, g Urea A1049, kg DTE BioChemica A1102, g DTT BioChemica A1101, g DTT - Solution (1 M) A3668, ml ß-Mercaptoethanol A1108, ml EDTA BioChemica A1103, kg EDTA disodium salt dihydrate BioChemica A1104, kg EDTA A5097, g EDTA-Solution ph 8.0 (0.5 M) A4892, ml EGTA für die Molekularbiologie A0878, g

7 Gel Filtration ReAction 7 Rapid Purification of Bio-Molecules Size Exclusion Chromatography (SEC, also referred to as Gel Filtration) is a chromatographic purification method for size-based separation of molecules in solution. Commonly this technique is used for large water soluble bio-molecules, such as DNA, oligonucleotides, proteins, antibodies and other polymers. AppliChem Panreac offers ready-to-use gravity flow and spin columns, and columns for FPLC systems, all products prepacked with Appli- Xchange. AppliXchange is a white solid composed of polymerized and cross-linked dextran: The beaded composite material is prepared in water or any desired aqueous buffer solution and subsequently cast into suitable columns. Molecules purified with AppliXchange are separated according to size. Smaller molecules pass significantly slower through the column than larger molecules. This is due to the longer path the smaller molecules must travel. This longer path arises from the pores of the beads. Due to the multitude of pores, most of the total volume of the beads is in fact water. Small mole cules enter the pores and get into the beads. The delay caused by entering the pores corre- lates with the molecular size. Molecules larger than the size exclusion cut-off (also referred to as molecular weight cut-off or size exclusion limit) cannot enter the pores and therefore flow around the porous beads rapidly passing the column. Absorption 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 Elution volume [ml] Elution profile for removal of excess FITC from IgG after coupling reaction. 1 mg IgG anti-rabbit and 0.1 µmol FITC in 1 ml DMSO/NaHCO 3 ; IgG (280 nm): black line; FITC (490 nm): magenta line.

8 8 ReAction Gel Filtration FAQs and Important Information What is the difference between G50 and G25? The number in the product description correlates to the swelling power of the matrix: Grade50 indicates that the dry matrix material will take up 5.0 x times its weight of water. G25 absorbs 2.5 x times its weight of water. The higher the swelling power the larger the pore size. The pore size determines the size exclusion cut-off. What does cut-off: 5 kd/10 bp mean? The cut-off gives the maximum molecule size that will be retained by the column. A size exclusion cut-off of 5 kda means, that molecules of a smaller size will remain (a certain time) in the matrix pores, while larger molecules rapidly run through the matrix. Why offering different particle sizes? The particle size or bead size does not influence the separation range or cut-off characteristics of the matrix, but is correlating to the flow rate. Small particles make the matrix more stable and are well suitable for centrifugation or vacuum. On the other hand, larger particle sizes enable adequate flow rates when using gravity based columns. The SEC matrix by scanning electron microscopy. AppliXchange G25-SF particles on a graphite surface.

9 Ready-to-Use Gel Filtration Columns and Plates DextraSEC and DextraSEP are ready-to-use gel filtration columns available in many different sizes (to process sample volumes from 10 µl up to 50 ml) and formats (gravity columns, spin columns, multiwell plates, FPLC columns) for convenient desalting and buffer exchange. Buffer conditions, ph value and temperature during the filtration process may be modified according to the needs of the molecules but not the column material. SEC can be performed in the presence of detergents, urea (up to 8 M) and guanidine hydrochloride (6 M), organic solvents (e.g. 24 % ethanol; 30 % propanol; 30 % acetonitrile), co-factors or ions, under basic and acidic conditions, high or low ionic strength. Gravity flow columns Column matrix: AppliXchange G25 M Cut off: > 5 kda;10 bp Bead size (dry): µm Hydrated gel filtration column for rapid and efficient removal of small molecules (salts, dyes, ammonia, haptens, biotin, etc.) from proteins, nucleic acids and other macromolecules. DextraSEC PRO (for protein purification) and DextraSEC NA (for nucleic acids) are available for processing sample sizes from 200 µl to 50 ml. FPLC columns Column matrix: AppliXchange G25 SF Cut off: > 5 kda; 10 bp Bead size (dry): µm Flow rate: 1 to 10 ml/min. Max. Backpressure: 3 bar NEW Hydrated gel filtration column for desalting, separa tion of larger bio-molecules (i.e. proteins such as anti bodies, enzymes or larger nucleic acids), and buffer exchange using Liquid Chromatography system. DextraSEP FPLC1 (A9790) and DextraSEP FPLC5 (A9749) can take maximum sample volumes of 0.3 and 1.5 ml. Multiwell plates Column matrix: AppliXchange G50 SF Cut off: > 25 kda; 20 bp Bead size (dry): µm Hydrated gel filtration multi-well plates for high-throughput applications (e.g. removal of excess Dye terminators from completed DNA sequencing reactions). DextraSEC 96W, DextraSEC 96W-large, and DextraSEC 384W are designed for sample volumes 15, 40, and 10 µl, respectively. Please visit our website

10 10 ReAction Gel Filtration Pre-packed and ready to go! Clean proteins or nucleic acids in 5 minutes only NEW 1. Spin column and discard storage buffer 2. Apply sample 3. Spin to elute purified sample into your preferred buffer solution Mini-spin columns for rapid desalting or buffer exchange Column matrix: AppliXchange G25 SF Cut off: > 5 kda; 10 bp Bead size (dry): µm Bed volume: 0.5 ml Sample volume: µl Description Order No. Content DextraSEC Mini-spinPRO Desalt (G-25) Purified proteins are eluted into pure water DextraSEC Mini-spinPRO Desalt (G-25), stabilized Purified Proteins are eluted into stabilized water DextraSEC Mini-spinPRO PBS (G-25) Elution into PBS, ph 7 DextraSEC Mini-spinPRO TRIS (G-25) Elution into ph 6 Tris buffer solution DextraSEC Mini-spinNA Desalt (G-25) Purified nucleic acids are eluted into pure water A9724, Columns A9708, Columns A8566, Columns A9692, Columns A9700, Columns Column matrix: AppliXchange G50 SF Cut off: > 25 kda; 20 bp Bead size (dry): µm Bed volume: 0.5 ml Sample volume: µl Description Order No. Content DextraSEC Mini-spinPRO Desalt (G-50) Purified proteins are eluted into pure water A9776, Columns DextraSEC Mini-spinPROPBS (G-50) Elution into PBS, ph 7 A9763, Columns DextraSEC Mini-spinPRO TRIS (G-50) Elution into Tris, ph 6 A9741, Columns DextraSEC Mini-spinNA Desalt (G-50) Purified nucleic acids are eluted into pure water A8563, Columns

11 a bit more ReAction 11 Antibiotics Amphotericin B BioChemica A1907, mg Ampicillin sodium salt BioChemica A0839, g Chloramphenicol BioChemica A1806, g Cycloheximide BioChemica A0879, g Doxycycline hyclate BioChemica A2951, g G418 disulfate BioChemica A2167, g G418 disulfate Solution, sterile A6798, ml Gentamycin sulfate BioChemica A1492, g Hygromycin B A5347, mg Hygromycin B - Solution A2175, ml Kanamycin sulfate BioChemica A1493, g Penicillin - Streptomycin (100X) Cell culture grade A8943, ml Penicillin G potassium salt BioChemica A1837, g Polymyxin B sulfate BioChemica A0890, g Spectinomycin dihydrochloride pentahydrate BioChemica A3834, g Streptomycin sulfate BioChemica A1852, g Vancomycin hydrochloride BioChemica A1839, g Salts & Buffers Magnesium chloride hexahydrate BioChemica A1036, g Sodium azide pure A1430, g Sodium chloride BioChemica A1149, kg Sodium chloride - Solution (5 M) A7006, L Sodium hydroxide pellets A6829, kg Potassium chloride BioChemica A1039, kg PBS tablets ph 7.4 (for 1 L) A9201, Tabs PBS tablets ph 7.2 (for 1 L) A9202, Tabs PBS buffer (1X, Dulbecco s) - Powder A0964, L Silver nitrate A3944, g Silver nitrate BioChemica A3972, g Tris A2264, kg Tris hydrochloride A3452, g TAE buffer (50X) A4686, L TBE buffer (10X) A3945, L Solvents Aceton BioChemica A3855,2500 2,5 L Dimethyl sulfoxide A3006, ml Ethanol absolute A3678, L Acetic acid 100 % BioChemica A3701,2500PE 2,5 L Formaldehyde - Solution 37 % A0877, ml Formamid BioChemica A0937,2500 2,5 L Glycerol 87 % BioChemica A0970, L Glycerol 87 % A3739, ml Methanol BioChemica A3493,2500PE 2,5 L 2-Propanol BioChemica A3465,2500 2,5 L 2-Propanol A3928,1000PE 1 L Hydrochloric acid (1 M) A6578, L Trichloroacetic acid - Solution 20 % BioChemica A0590, ml Water bidistilled, sterile A4042,0500 A4042, ml 1 L

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