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1 E N Z Y M E S F O R M O L E C U L A R B I O L O G Y

2 More space,more products Nun ist es vollbracht. GeneCraft firmiert unter einer neuen Adresse. Das großräumige Gebäude in Lüdinghausen eröffnet unzählige Möglichkeiten die umfangreiche Produktpalette den Anforderungen unserer Kunden anzupassen. Stellen Sie uns auf die Probe. Sollten Sie Fragen zu unseren Produkten oder Anregungen zur Erweiterung unseres Kataloges haben, so stehen wir ihnen gerne zur Verfügung. GENECRAFT GmbH Raiffeisenstr Lüdinghausen

3 TABLE OF CONTENTS 3 Summary of our thermostable polymerases 4 BioTherm DNA polymerase 5 First description of Taq polymerase 7 BioThermMix 8 NeoTherm DNA polymerase 10 SupraTherm DNA polymerase 11 CoverIt overlay for PCR 12 PCR-Grade H 2 O 12 Crystal violet buffer 13 BioThermRed DNA polymerase 14 BioThermT DNA polymerase 15 BioThermBio DNA polymerase 17 BioThermStar DNA polymerase 18 BioThermStarMix DNA polymerase 20 BioThermAB DNA polymerase 22 Hot Start Taq monoclonal antibody 23 Hot Start PCR compound 24 KlenTherm DNA polymerase 25 KlenThermN DNA polymerase 28 KlenThermPlatinum DNA polymerase 29 KlenThermase DNA polymerase 31 KlenThermaseN DNA polymerase 32 DNA cycle sequencing kit 32 Tth inorganic pyrophosphatase 34 Sequencing and pyrophosphatase 35 AccuTherm DNA polymerase 36 Comparison of some thermostable polymerases 37 Synergy DNA polymerase 38 SynergyN DNA polymerase 39 SynergyT DNA polymerase 40 SynergyPlus DNA polymerase 41 TthPlus DNA polymerase 42 Comparison of TthPlus and GeneScript RT 44 GeneScript reverse transcriptase 48 RT-PCR using GeneScript reverse transcriptase 48 RNase-Inhibitor 49 Klenow fragment 50 T4 polynucleotide kinase 51 T4 DNA ligase 52 Tth DNA ligase 53 SP6 RNA polymerase 54 T7 RNA polymerase 54 Restriction enzymes 55 Random prime labeling kit 58 dntp set 59 DNA polymerization mix 20 & dntps 60 dntp custom service 61 Biotin-4-dUTP 61 Biotin-11-dUTP 61 Uracil-DNA glycosylase 62 λ DNA 62 T-Vector System 63 pbs-ks-ecorv 66 BstE II-λ-DNA marker 66 Hpa II-pBS-DNA marker 67 EcoRI-λ-DNA marker 68 HindIII-λ-DNA marker 69 EcoRI+HindIII-λ-DNA marker bp ladder 71 1 kb ladder 72 MegaPure DNA purification kit 73 Treponema pallidum recombinant antigen- pa 74 Monoclonal IgG to Thermus aquaticus DNA-polymerase 74 IPTG (Isopropyl-ß-D-thiogalactoside) 75 Acrylamid solutions 76 Laemmli puffer 78 TBE buffer 78 TEMED 79 Amoniumpersulfate 79 Agarose LSL Agarose S Agarose S-IM Bounded ethidium bromide agarose 83 Magnetic particles 84 MagicTubes for perfect PCR 85 Laboratory consumables 88 Index 89 Order-form 90 That s new 91

4 SUMMARY OF OUR THERMOSTABLE POLYMERASES 4 CAT. NO. NAME ORGANISM APPLICATION REACTION BUFFERS GC-004 AccuTherm P. furiosis DNA polymerase with high fidelity PCR DNA polymerase proof-reading activity GC-002 BioTherm * T. aquaticus DNA polymerase for PCR DNA polymerase robust amplifications GC-008 BioThermAB T. aquaticus mixture of BioTherm and Hot-Start PCR DNA polymerase Hot-Start Antibody PCR with high specificity GC-047 BioThermMix T. aquaticus ready-mix for High through-put PCR amplifications routine diagnostic PCR GC-041 BioThermStarMix T. aquaticus Hot-Start ready-mix for Hot-Start high amplifications through-put PCR GC-021 BioThermRed T. aquaticus red-dyed BioTherm PCR DNA polymerase GC-045 BioThermStar T. aquaticus chemically modified form Hot-Start PCR DNA polymerase of BioTherm GC-001 KlenTherm T. aquaticus Klenow fragment Specific PCR DNA polymerase of BioTherm GC-018 KlenThermase T. aquaticus modified KlenTherm DNA dideoxysequencing DNA polymerase GC-023 KlenThermN T. aquaticus modified KlenTherm PCR of long and DNA polymerase GC-rich fragments GC-043 KlenThermaseN T. aquaticus modified KlenThermase Sequencing of long DNA polymerase and GC-rich fragments GC-046 KlenThermPlatinum T. aquaticus modified KlenTherm PCR with excellent DNA polymerase specificity GC-031 NeoTherm * T. aquaticus DNA polymerase for PCR DNA polymerase robust amplifications GC-022 SupraTherm * T. aquaticus DNA polymerase for PCR DNA polymerase standard amplifications GC-005 Synergy Mixture mixture of KlenTherm Long and accurate DNA polymerase and AccuTherm PCR GC-028 SynergyN Mixture mixture of KlenThermN Long and accurate DNA polymerase and AccuTherm fragments PCR of GC-rich GC-048 SynergyPlus Mixture Taq- and pyrophospha- Very long-range PCR DNA polymerase tase based mixture GC-049 SynergyT Mixture mixture of Synergy Low-specificity PCR for DNA polymerase with TthPlus degenerated primer GC-003 TthPlus T.thermophilus Tth DNA polymerase with RT-PCR DNA polymerase reverse transcriptase activity 5x improved buffer: 350 Tris-HCl ph 8,8 83 mm (NH) 4 SO 4, 100 mm KCl 22 mm MgCl 2 0,75% Triton X100, 10x buffer: 200 Tris-HCl ph 8,5 100 mm (NH) 4 SO 4 20mM MgCl 2 1% Triton X100,10 x buffer can be modified by adding KCl and MgCl 2 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 8,8 0,1%Tween 20, 15 mm MgCl 2 10x reaction buffer without MgCl 2 is available 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 8,8 0,1%Tween20, 15 mm MgCl 2,10x reaction buffer without MgCl 2 is available upon request BioThermMix contains already 2,5x reaction buffer, dntps and BioTherm DNA polymerase ready for PCR BioThermStarMix contains already 2.5x reaction buffer, dntps and BioThermStar DNA polymerase ready for Hot-Start PCR 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 8,3 0,1%Tween20, 15 mm MgCl 2 10x reaction buffer without MgCl 2 is available upon request 10x buffer: 500 mm KCl, 100 mm Tris-HCl ph 9 1% Triton X100 add MgCl 2 to a final concentration of 3,5 mm 10x buffer: 500 mm KCl, 100 mm Tris-HCl ph 9 1% Triton X100 add MgCl 2 to a final concentration of 3,5 mm 10x buffer: 500 mm KCl, 100 mm Tris-HCl ph 9 1% Triton X100 add MgCl 2 to a final concentration of 3,5 mm 10x buffer: 500 mm KCl, 100 mm Tris-HCl ph 9 1% Triton X100 add MgCl 2 to a final concentration of 3,5 mm 10x buffer: 500 mm KCl, 100 mm Tris-HCl ph 9 1% Triton X100 add MgCl 2 to a final concentration of 3,5 mm 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 8,8 0,1%Tween20, 15 mm MgCl 2 10x reaction buffer without MgCl 2 is available upon request 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 8,8 0,1%Tween20, 15 mm MgCl 2 10x reaction buffer without MgCl 2 is available upon request 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 9,1 8% glycerol, 2% DMSO, 35 mm MgCl 2 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 9,1 8% glycerol, 2% DMSO, 35 mm MgCl 2 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 9,1 8% glycerol, 2% DMSO, 35mM MgCl 2 10x buffer: 160 mm (NH) 4 SO mm Tris-HCl ph 9,1 8% glycerol, 2% DMSO, 35 mm MgCl 2 10x one-tube buffer: 500 mm bicine-koh ph 8,3 1 M KOAc ph7,5 30% glycerol (v/v); extra solution: 50 mm Mn(OAc) 2, 10x RT buffer: 166 mm (NH) 4 SO mm Tris-HCl ph 8,8 0,1%Tween20; extra solution: 25 mm MnCl 2 5x PCR buffer: 83 mm (NH) 4 SO mm Tris-HCl ph 8,8 0,1%Tween20, 3,75 mm EGTA, 25% glycerol (v/v); extra solution: 50 mm MgCl 2 *BioTherm, NeoTherm and SupraTherm are comparable TaqDNA polymerases isolated by slightly different procedures

5 BioTherm DNA POLYMERASE BioTherm DNA polymerase is a thermostable DNA - polymerase purified from the Thermus aquaticus strain in accordance with the procedures developed by Kaledin (1,2,3) for the isolation of thermostable enzymes processing DNA polymerase activity from thermophilic bacteria. Amplification of DNA fragments (100 bp to 10kb) can be achieved with this enzyme. The enzyme has both 5'-3' polymerase- and 5'-3' exonuclease activities. BioTherm can add a single template-directed deoxyadenosin (A) residue to the 3 end of duplex PCR products. This property allows easy and efficient ligation of PCR products in TA cloning vectors. BioTherm DNA-Polymerase ist eine hitzestabile DNA- Polymerase, welche nach dem Protokoll von Kaledin (1,2,3) zur Reinigung von hitzestabilen Enzymen mit Polymerase-Aktivität aus thermophilen Bakterien aus Thermus aquaticus isoliert wurde. Diese Polymerase ist in der Lage DNA-Fragmente mit einer Länge von 100 bp bis 10 kb zu amplifizieren. BioTherm DNA-Polymerase besitzt sowohl eine 5-3 Polymerase- als auch eine 5-3 Exonuklease-Aktivität. Das Enzym kann einen einzelnen, Template-abhängigen Deoxyadenosin (A) Rest an das 3 -Ende doppelsträngiger PCR-Produkte anhängen. Diese Eigenschaft erlaubt die einfache und effiziente Ligation von PCR-Produkten in TA- Klonierungs-Vektoren. 5 APPLICATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acidinsoluble form in 30 minutes at 72ºC under the assay conditions (25 mm TAPS (tris-(hydroxy-methyl)-methyl-aminopropanesulfonic acid, sodium salt) ph 9.3 (at 25ºC); 50 mm KCl; 2 mm MgCl 2 ; 1 mm ß-mercaptoethanol) and activated calf thymus DNA as substrate. STORAGE BUFFER 10 mm K-phosphate buffer ph 7.0, 100 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 0.01% Tween 20, 50% glycerol (v/v) STORAGE TEMPERATURE Store BioTherm DNA polymerase below 0ºC, preferably at -20ºC, in a constant temperature freezer. STORAGE TEMPERATURE 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.8 (at 25ºC), 15 mm MgCl 2, 0.1% Tween 20 The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. 10X REACTION BUFFER 1.5 ml 10x reaction buffer (contains 15 mm MgCl 2 ) Cat. No. GC ml 10x reaction buffer without MgCl 2 plus 50 mm MgCl 2 separately Cat. No. GC ASSOCIATED ACTIVITIES Endonuclease- and exonuclease activities were not detectable after 2 and 1 hours incubation, respectively, of 1 µg lambda DNA and 0.22 µg of EcoRI digested lambda DNA, respectively, at 72ºC in the presence of units of BioTherm DNA polymerase. COMPANION PRODUCTS BioThermRed DNA polymerase, SupraTherm DNA polymerase, dntps, T-Vector GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

6 AMPLIFICATION CONDITIONS 10x reaction buffer 3 µl dntps (200 µm each) 5 µl human genomic DNA ( ng) 1 µl forward primer (25 pm) 2 µl reverse primer (25 pm) 2 µl BioTherm (0.2-1 units/µl) 1 µl H 2 O 16 µl 58ºC 0.5 min 72ºC 4 min 93ºC 20 sec 30 cycles total 30 µl 6 REFERENCES 1 Kaledin, A.S., et al. (1980) Biokhimiya 45, Kaledin, A.S., et al. (1981) Biokhimiya 46, Kaledin, A.S., et al. (1982) Biokhimiya 47, 1785 COMPARISON OF DIFFERENT TAQ DNA POLYMERASES MIXTURE Template 1 µl (IN4/TNOMF5-product) Buffer 4 µl dntps 8 µl => 200 µm MgCl µl (lanes 3-6) => 1.5 mm IN4 2 µl TNOMF5 2 µl (lanes 1,3,5,7) TNOMF6 2 µl (lanes 2,4,6,8) Polymerase 2 µl H 2 O added to 30 µl Program 1 94 C 2 min 2 94 C 30 sec 65 C 30 sec 30 cycles 72 C 30 sec (75 C by Eurogentec HA DNA polymerase) 3 72 C 7 min 4 4 C POLYMERASES Lane Marker 9 Marker GEL 1.5% Agarose-TAE-Gel, 85V 10 µl of each mixture per lane 1+2 Sigma, 1 u 3+4 Promega, 1 u 5+6 Eurogentec HA, 0.3 u 7+8 BioTherm, 1 u bp 240 bp

7 THE FIRST OF TAQ POLYMERASE by the Russian scientist Kaledin 7 Kaledin, A.S, at al. (1980) Biokhimia 45, 494

8 8 BioThermMix BioTherm Mix is a 2.5x concentrated reagent mix for PCR comprising BioTherm DNA polymerase (0.06 u/µl), 2.5x PCR-Buffer with 3.75 mm MgCl 2, 500 µm of each dntp and stabilizers. The 2.5x concentration of the Mix provides high versatility for settting-up PCR reactions. Up to 60% of the final reaction volume can be used for the addition of primer and template DNA solutions, co-solvents or other additives, if necessary. BioThermMix ist ein 2.5x konzentrierter Reagenzien-Mix für PCR-Reaktionen. Der Mix enthält Bio- Therm DNA-Polymerase (0.06 u/µl), 2.5x PCR- Puffer mit 3.75 mm MgCl 2, 500 µm von jedem dntp und stabilisierende Substanzen. Die 2.5x Konzentration des BioThermMix ermöglicht einen flexiblen Einsatz in den PCR-Reaktionen.Auf diese Weise kann bis zu 60 % des endgültigen Reaktion-Volumens für die Zugabe von Primern, Template-DNA, sowie anderen Reaktionssubstanzen genutzt werden. APPLICATION BioTherm Mix is suitable and tested for amplification of genomic targets ranging from l00 bp to 4 kb and of episomal targets (lambda phage; plasmids) up to 10 kb under various reaction conditions. high through-put PCR routine diagnostic PCR requiring high reproducibility DNA sequencing template preparation STORAGE CONDITIONS BioTherm Mix can be stored at either -20 C in a freezer or at 2-8 C in a usual refrigerator. Shipment at ambient temperature is possible without reduction of PCR performance and activity. Storage at 2-8 C is convenient for easy and time-saving assembling of the PCR assays. Storage of BioTherm Mix at -20 C is recommended for long-term storage after the mix has been used once under non-sterile conditions. Multiple freezing and thawing do not affect the performance or activity of the Mix. At 2-8 C the BioTherm Mix is at least stable for 12 months, in frozen state for 2 years. NOTE Do not contaminate the BioTherm Mix with primers and template DNA used in individual reactions. Thaw and mix all components thoroughly, spin down shortly and chill on ice. It is very important to mix the BioTherm Mix before use to avoid localized concentration! PROTOCOL 1 Place the PCR tubes on ice. 2 Prepare first a template/primer mix according to the volumes given in the table below for different reaction volumes. Mix the template/primer mix and chill on ice. 3 Dispense now the corresponding volume of Bio- Therm Mix (20 µl for a 50 µl reaction) followed by the template/primer mix into each reaction tube. Close tubes and mix well. Spin down shortly, if necessary, and chill the tubes on ice. 4 Start the program on the thermal cycler. Transfer the tubes directly from ice into the thermal cycler when the temperature of the block has reached 90 C. IMPORTANT NOTE The stabilizers in the BioTherm Mix are potential growth substrates for bacterial contaminations! If the Mix has been opened and used under non-sterile conditions, the residual moiety should be used during the next 2 weeks with intermediate storage at 2-8 C or should be frozen at -20 C for longer storage!

9 BioThermMix PCR Sterile redi- Sense Antisense Template 2.5x PCR Volume stilled H 2 O Primer Primer DNA Mix 100 µl up to 60 µl x µl y µl z µl 40 µl 50 µl up to 30 µl x µl y µl z µl 20 µl 25 µl up to 15 µl x µl y µl z µl 10 µl 20 µl up to 12 µl x µl y µl z µl 8 µl Final concentrations: 200 nm 200 nm ng 1x 9 Other variable reaction conditions (temperatures, cycling times, concentrations of template, primers, magnesium and polymerase) have to be optimized empirically for each template/primers combination. Most PCR applications work at the standard concentration of 1,5 mm Mg 2+ provided with 1x diluted PCR Mix. Optimal Mg 2+ concentration higher than 1,5 mm can be adjusted using a separate magnesium solution or by increasing stepwise (5 µl increments) the amount of BioThermMix added to the reaction assay. This approach can also be used to improve the product yield in amplification of difficult targets on complex template DNA (see table below). Optimization by adding variable amounts of concentrated BioThermMix to a 50 µl-assay: Volume of Volume of Final Mg 2+ Final dntp- Taq units Final 2,5x PCR template/ Concen- Concen- per concen- Mix primer mix tration tration reaction trations 20 µl 30 µl 1.5 mm 200 µm 1.25 u 1x 25 µl 25 µl 2.0 mm 250 µm 1.6 u 1.25x 30 µl 20 µl 2.25 mm 300 µm 1.9 u 1.5x GC ml

10 10 NeoTherm DNA POLYMERASE NeoTherm is a thermostable DNA polymerase purified from the Thermus aquaticus strain in accordance with the procedures developed by Kaledin for the isolation from thermophilic bacteria of thermostable enzymes processing DNA polymerase activity. It is a highly processive 5'-3' DNA polymerase lacking 3'-5'-exonuclease activity. The enzyme is highly purified and is free of nonspecific endo- or exonucleases. This polymerase has a template-dependent activity which adds a single deoxyadenosine (A) to the 3' ends of PCR products (3'-A-overhangs). This property allows easy and efficient ligation of PCR products in TA cloning vectors. NeoTherm DNA-Polymerase ist eine hitzestabile DNA-Polymerase, welche nach dem Protokoll von Kaledin (1,2,3) zur Reinigung von hitzestabilen Enzymen mit Polymerase-Aktivität aus thermophilen Bakterien aus Thermus aquaticus isoliert wurde. Dies ist eine äußerst aktive 5-3 DNA-Polymerase ohne 3-5 Exonuklease-Aktivität. Das Enzym weist einen sehr hohen Reinheitsgrad auf und enthält daher keinerlei unspezifische Endo- oder Exonukleasen. Die Polymerase besitzt eine Template-abhängige Aktivität, welche einen einzelnen Deoxyadenosin (A) -Rest an das 3 -Ende von PCR-Produkten anhängt (3 -A- Überhänge). Diese Eigenschaft erlaubt die einfache und effiziente Ligation von PCR-Produkten in TA-Klonierungs- Vektoren. APPLICATION PCR DNA labelling CONCENTRATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acid-insoluble form in 30 min at 72 C. STORAGE BUFFER 20 mm Tris-HCl ph 8.0, 100 mm KCl, 0.1 mm EDTA, 5 mm DTT, 50% glycerol, 1% Triton X-100 STORAGE TEMPERATURE -20 C 10X REACTION BUFFER 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.8 (at 25ºC), 15 mm MgCl 2, 0.1% Tween 20 The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. QUALITY CONTROL Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases The enzyme is also tested to verify that less than 10 copies of bacterial 16S ribosomal RNA gene sequences are present in a standard 2.5 unit aliquot. GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

11 SupraTherm DNA POLYMERASE SupraTherm DNA polymerase is a thermostable DNA polymerase purified from the Thermus aquaticus strain by several rounds of liquid chromatography. The purity of SupraTherm DNA polymerase is more than 90% of the total protein in the preparation. Amplification of DNA fragments (100 bp to 5 kb) can be achieved with it. The enzyme has both, 5'-3' polymeraseand 5'-3'exonuclease activities. SupraTherm can add a single template-directed deoxyadenosin (A) residue to the 3 end of duplex PCR products. This property allows easy and efficient ligation of PCR products in TA cloning vectors. 11 SupraTherm DNA-Polymerase ist eine hitzestabile DNA-Polymerase, die in mehreren Durchläufen mittels Flüssigkeits-Chromatographie aus Thermus aquaticus isoliert wurde. Der Reinheitsgrad der SupraTherm DNA-Polymerase beträgt mehr als 90 % bezogen auf den Gesamt-Proteingehalt. Das Enzym ist in der Lage DNA-Fragmente mit einer Länge von 100 bp bis 5 kb zu amplifizieren. SupraTherm DNA-Polymerase besitzt sowohl eine 5-3 Polymerase- als auch eine 5-3 Exonuklease-Aktivität. Das Enzym kann einen einzelnen, Template-abhängigen Deoxyadenosin (A) Rest an das 3 -Ende doppelsträngiger PCR-Produkte anhängen. APPLICATION The enzyme may be used for PCR, thermosequencing and for other research and diagnostic applications. SupraTherm DNA polymerase is the best enzyme for everyday routine applications. The enzyme has limited application for PCR with low-abundance template (less then 100 DNA molecules of template). Please use for PCR of low-abundance templates and for other more sophisticated techniques BioTherm DNA polymerase. CONCENTRATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acid-insoluble form in 30 minutes at 72ºC under the assay conditions (25 mm TAPS (tris-(hydroxy-methyl)-methyl-aminopropanesulfonic acid, sodium salt) ph 9.3 (at 25ºC), 50 mm KCl, 2 mm MgCl 2, 1 mm ß-mercaptoethanol) and activated calf thymus DNA as substrate. STORAGE BUFFER 10 mm K-phosphate buffer ph 7.0, 100 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 0.01% Tween 20; 50% glycerol (v/v) STORAGE TEMPERATURE Store SupraTherm DNA polymerase below 0ºC, preferably at -20ºC, in a constant temperature freezer. 10X REACTION BUFFER 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.8 (at 25ºC), 15 mm MgCl 2, 0.1% Tween 20 The 10x reaction buffer(on request with or without MgCl 2 ) is delivered free of charge. 1.5 ml 10x reaction buffer (contains 15 mm MgCl 2 ) Cat. No. GC ml 10x reaction buffer without MgCl 2 plus 50 mm MgCl 2 separately Cat. No. GC QUALITY CONTROL SupraTherm DNA polymerase was tested for the absence of unspecific endo- and exonucieases activities. COMPANION PRODUCTS BioThermRed DNA polymerase, BioTherm DNA polymerase, dntps, T-Vector GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

12 CoverIT OVERLAY FOR PCR REACTIONS 12 CoverIt can be used as a sample overlay in PCR reaction tubes. The use of CoverIt prevents evaporation and subsequent condensation of the reaction mixture in the reaction tube. Upon cooling to +4 o C CoverIt becomes solid allowing convenient recovering of the reaction mix by sticking through it with a pipet tip. STORAGE TEMPERATURE store at room temperature CoverIt kann als Proben-Überschichtung in PCR Reaktions-Gefäßen verwendet werden. Die Verwendung von CoverIt verhindert die Verdunstung und anschließende Kondensation des Reaktionsansatzes im Reaktionsgefäß. Nach Kühlung auf +4 C wird CoverIt fest, so dass man mit einer Pipettenspitze durchstechen kann, um den Reaktionsansatz heraus zu pipettieren. PROTOCOL For µl PCR mix use 60 µl (two drops) CoverIt. GC ml PCR-GRADE H 2 O GC-058 1,5 ml

13 CRYSTAL VIOLET BUFFER By including crystal violet in the gel and loading bufffer it is possible to visualize DNA bands as they separate during agarose gel electrophoresis. This is particularly useful for isolating DNA fragments for cloning work. The advantage of this method is that the DNA is not exposed to the damaging effects of ultraviolet (UV) light, as occurs when ethidium bromide is used for staining. The improvement in cloning efficiency observed when crystal violet is used is threefold when compared with ethidium bromide and a transilluminator with max. 320 nm and tenfold in comparison with a shorter wavelength (302 nm). APPLICATION Cloning of DNA fragments (PCR products and DNA molecules digested by restriction enzymes) STORAGE TEMPERATURE +4 C to -20 C REFERENCES Rand, K. N. (1996) Crystal violet can be used to visualize DNA bands during gel electrophoresis and to improve cloning efficiency. Elsevier Trends Journals Technical Tips Online, T Mit Hilfe des Crystal Violet in Gel und Puffer ist es möglich die DNA-Banden sichtbar zu machen, während sie sich in einer Agarose-Gel-Elektrophorese trennen. Dies ist besonders nützlich bei DNA-Fragmenten, die für Klonierungen isoliert werden. Der Vorteil dieser Methode ist, dass die DNA nicht den schädigenden Einflüssen ultravioletten (UV) Lichts ausgesetzt werden muss, was nötig ist, wenn Ethidiumbromid für die Färbung verwendet wird. Der Erfolg einer Klonierung ist nach Verwendung von Crystal Violet dreimal höher als bei Verwendung von Ethidiumbromid oder einem Transilluminator mit max. 320 nm, sowie zehnmal höher bei einer kürzeren Wellenlänge (302 nm). PROTOCOL Crystal violet has a lower sensitivity than ethidium bromide. Load as much as 1 to 3 µg DNA in a slot. If less than 100 ng of the required fragment is loaded onto the gel it may be necessary to make a control with ethidium bromide by running a second minigel at the same time. Agarose should be prepared by adding 100 µl Crystal violet gel buffer to 100 ml gel. Add 3-5 µl Crystal Violet loading buffer to 30 µl digest or PCR aliquot and load that into the slot. For maximum sensitivity the running buffer should only just cover the gel. After electrophoresis, place the gel on a light box to visualize separated fragments. Crystal violet is runnning in opposite direction as DNA, so do not run the gel for a too long time. 13 GC µl loading buffer,10 ml gel buffer (100 rxns).

14 14 BioThermRed DNA POLYMERASE BioThermRed DNA polymerase is a mixture of Bio- Therm DNA polymerase and red pigment that works like BioTherm. After addition of BioTherm- Red a thin red layer on the bottom of the tube appears. So you have a visible pipetting control. Furthermore you can see, if your reaction is already mixed, when the color of your reaction becomes uniform. BioThermRed ist eine neue, mit rotem Farbstoff versehene Variante der BioTherm DNA Polymerase. Sie funktioniert genauso wie BioTherm DNA Polymerase. Nach Zugabe von BioThermRed erscheint eine dünne rote Schicht am Boden des Reaktionsgefäßes. Dadurch haben Sie eine deutlich sichere Pipettierkontrolle. Weiterhin können Sie anhand der roten Farbe erkennen, ob ihr Ansatz gut durchmischt ist. Gleichmäßige Farbverteilung deutet auf eine vollkommene Durchmischung der Probe hin. CONCENTRATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acidinsoluble form in 30 minutes at 72ºC under the assay conditions (25 mm TAPS (tris-(hydroxy-methyl)-methyl-aminopropanesulfonic acid, sodium salt) ph 9.3 (at 25ºC), 50 mm KCl, 2 mm MgCl 2, 1 mm ß-mercaptoethanol) and activated calf thymus DNA as substrate. STORAGE BUFFER 10 mm K-phosphate buffer ph 7.0, 100 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 0.01% Tween 20,0.2% indicator red dye, 50% glycerol (v/v) The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. STORAGE TEMPERATURE Store BioThermRed DNA polymerase below 0ºC, preferably at -20ºC, in a constant temperature freezer. 10X REACTION BUFFER 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.8 (at 25ºC), 15 mm MgCl 2, 0.1% Tween ml 10x reaction buffer (contains 15 mm MgCl 2 ) Cat. No. GC ml 10x reaction buffer without MgCl 2 plus 50 mm MgCl 2 separately Cat. No. GC ASSOCIATED ACTIVITIES Endonuclease- and exonuclease activities were not detectable after 2 and 1 hours incubation, respectively, of 1 µg lambda DNA and 0.22 µg of EcoRI digested lambda DNA, respectively, at 72ºC in the presence of units of BioThermRed DNA polymerase. COMPANION PRODUCTS BioTherm DNA polymerase, SupraTherm DNA polymerase, dntps GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

15 BioThermT DNA POLYMERASE BioThermT is a modified BioTherm DNA polymerase to facilitate incorporation of Biotin-dUTP and Digoxigenin-dUTP in DNA. BioThermT ist eine modifizierte BioTherm DNA- Polymerase, die es ermöglicht Biotin-dUTP und Digoxigenin-dUTP in DNA einzubringen. 15 CONCENTRATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acidinsoluble form in 30 minutes at 72ºC under the assay conditions (25 mm TAPS (tris-(hydroxy-methyl)-methyl-aminopropanesulfonic acid, sodium salt) ph 9.3 (at 25ºC), 50 mm KCl, 2 mm MgCl 2, 1 mm ß-mercaptoethanol) and activated calf thymus DNA as substrate. STORAGE BUFFER 10 mm K-phosphate buffer ph 7.0, 100 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 0.01% Tween 20,0.2% indicator red dye, 50% glycerol (v/v) The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. STORAGE TEMPERATURE Store BioThermT DNA polymerase below 0ºC, preferably at -20ºC, in a constant temperature freezer. 10X REACTION BUFFER 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.8 (at 25ºC), 15 mm MgCl 2, 0.1% Tween ml 10x reaction buffer (contains 15 mm MgCl 2 ) Cat. No. GC ml 10x reaction buffer without MgCl 2 plus 50 mm MgCl 2 separately Cat. No. GC ASSOCIATED ACTIVITIES Endonuclease- and exonuclease activities were not detectable after 2 and 1 hours incubation, respectively, of 1 µg lambda DNA and 0.22 µg of EcoRI digested lambda DNA, respectively, at 72ºC in the presence of units of BioThermT DNA polymerase. COMPANION PRODUCTS BioTherm, BioThermBio A 700 bp DNA fragment of a single copy gene was amplified without (lane 1) and with the addition of biotin-11-dutp using different concentrations (lanes 2-4, MW = molecular weight standard). PCR reactions were run for 40 cycles, 10 sec at 92 C,15 sec at 65 C, and 2 min at 72 C in a volume of 25 µl, including 10 x amplification buffer (Genecraft), 2 mm MgCl 2,5 pmol of each forward and reverse primers, 1 ng of DNA, 1 unit of BioTherm T Taq polymerase (Genecraft) and 0.1 mm of each dntps, whereas dttp was proportionally substituted with increasing concentrations of biotin-11-dutp: 20% (lane 2), 50% (lane 3), and 70% (lane 4). The incorporation of biotin-11-dutp correlates with a size shifting of the amplified product. Note, the decrease of PCR efficiency at a proportion of 70% biotin -11-dUTP (lane 4) compared to the lower concentrations. MW MW

16 BioThermT DNA POLYMERASE pg 50pg 5pg 20% bio-dutp 50% bio-dutp 70% bio-dutp A 700 bp DNA fragment was internally labeled by PCR mediated incorporation of biotin-11-dutp (biotin-11- dutp/dttp = 1/4, 1/1, and 2.3, respectively; see figure 1). The PCR products were diluted to 500 pg, 50 pg, and 5 pg and dot blotted onto nylon membrane. The DNA was cross linked to the membrane by UV radiation and visualized by colorimetric detection by means of Streptavidin/alkaline phosphatase conjugate incubation in the presence of BCIP and XPhosphat for 3 hours at 37 C. Signal intensity was strongest at 1/1 concentration (50%) of biotin-11-dutp. The efficiency of PCR mediated incorporation of biotin- 11-dUTP into a DNA fragment of 900 bp was assesed quantitatively and by comparing different Taq polymerases using a ratio of biotin-11-dutp/dttp of 1/1 (amplification conditions as described in figure 1). Lane 1 shows the unincorporated PCR product, lanes 2-4 show the biotinylated products using 0.1, 1, and 10 ng of starting DNA template and BioTherm T polymerase (Gencraft), lane 5 and 6 depicts biotinylated products using regular BioTherm polymerase (Genecraft) and an enzyme from a competitor, respectively. Note, the template dependent increase of PCR product yield, and the high efficiency of both BioTherm polymerases compared to a competitor s polymerase. MW MW GC GC GC GC u 250 u 500 u 1000 u

17 BioThermBio DNA POLYMERASE BioThermBio is a novel thermostable DNA polymerase that dramatically improves incorporation of Biotin- and Digoxigenin-dUTPs as compared to Taq DNA polymerase. BioThermBio ist eine neue hitzestabile DNA-Polymerase, welche das Einbringen von Biotin- und Digoxigenin-dUTPs in DNA grundlegend verbessert. 17 Biotine-11-dUTP labelling of PCR DNA fragment (650 bp) by thermophilic DNA polymerases. Lane 1 Amplification by Taq polymerase, dntps 200mM each, no Biotine-11-dUTP Lane 2 Amplification by Taq polymerase, datp, dgtp, dctp 200 mm each, dttp 130 mm, Biotin-11-dUTP 70 mm. Lane 3 Amplification by NEW polymerase BioTherm- Bio, datp, dgtp, dctp 200 mm each, dttp 130 mm, Biotin-11-dUTP 70 mm. Analysis with streptavidin/- alcalic phosphatase conjugates shows that specific incorporation of Biotin-11-dUTP was 20 times higher with NEW polymerase. 900 dp 800 dp 700 dp 600 dp M GC GC GC GC u 250 u 500 u 1000 u

18 18 BioThermStar DNA POLYMERASE BioThermStar is a thermostable DNA polymerase purified from the Thermus aquaticus strain in accordance with the procedures developed by Kaledin for the isolation of thermostable enzymes processing DNA polymerase activity from thermophilic bacteria. Bio- ThermStar is a modified form of the enzyme designed for Hot-Start-PCR. It is a highly processive 5'-3' DNA polymerase lacking 3'-5'-exonuclease activity. The enzyme is highly purified and is free of nonspecific endo- or exonucleases. BioThermStar ist eine hitzestabile DNA-Polymerase, welche nach dem Protokoll von Kaledin (1,2,3) - zur Reinigung von hitzestabilen Enzymen mit Polymerase- Aktivität aus thermophilen Bakterien - aus Thermus aquaticus isoliert wurde. Diese 5-3 DNA-Polymerase ist eine modifizierte Form des Enzyms und für Hot- Start-PCR-Reaktionen entwickelt worden. Sie besitzt keine 3-5 Exonuklease-Aktivität. Das Enzym weist einen sehr hohen Reinheitsgrad auf und enthält daher keinerlei unspezifische Endo- oder Exonukleasen. Comparison of BioThermStar (GeneCraft) with AmpliTaqGold (PE) BioThermStar units pre-heating-time UNIT DEFINITION One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dntp into acid-insoluble material in 30 min at 72 C. STORAGE BUFFER 10 mm K-phosphate buffer ph 7.0, 100 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 0.01% Tween 20, 50% glycerol (v/v) BUFFER PH We strongly recommmend to use buffers with ph 8.3! AmpliTaqGold ACTIVATION This polymerase is inactive until incubated 7-10 min at 95 C! These activation conditions are extremely important! The activation completely prevents nonspecific primer annealing and the formation of primer-dimers during setup. REACTION TEMPERATURE It is also very important to pay great attention to the actual temperature (95 C) inside the tube! APPLICATION Hot-Start-PCR PCR with high specificity CONCENTRATION 5 units/µl STORAGE TEMPERATURE -20 C 10X REACTION BUFFER 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.3 (at 25 C), 15 mm MgCl 2, 0.1% Tween 20 The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. Please note the difference between BioTherm and BioThermStar buffers! QUALITY CONTROL Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases. COMPANION PRODUCTS BioThermAB, MagicTubes

19 BioThermStar DNA POLYMERASE BioThermStar IN A TAQMAN ASSAY 200 nm dntp s 500 nm Primer 1.25 u BioTherm Star/0.25 µl 3 mm MgCl2 300 nm passive reference dye (Rox) 100 nm TaqMan-probe 19 GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

20 20 BioThermStarMix BioThermStarMix is a 2.5x concentrated reagent mix for Hot Start PCR comprising BioThermStar DNA polymerase (0.06 u/µl), 2.5x PCR-Buffer with 3.75 mm MgCl 2, 500 µm of each dntp and stabilizers. The 2.5x concentration of the Mix provides high versatility for setting-up Hot Start PCR reactions. Up to 60% of the final reaction volume can be used for the addition of primer and template DNA solutions, co-solvents or other additives, if necessary. BioThermStarMix ist ein 2.5x konzentrierter Reagenzien-Mix für Hot Start PCR-Reaktionen. Der Mix enthält BioThermStar DNA-Polymerase (0.06 u/µl), 2.5x PCR-Puffer mit 3.75 mm MgCl 2, 500 µm von jedem dntp und stabilisierende Substanzen. Die 2.5x Konzentration des BioThermStarMix ermöglicht einen flexiblen Einsatz in den Hot Start PCR-Reaktionen. Auf diese Weise kann bis zu 60 % des endgültigen Reaktion-Volumens für die Zugabe von Primern, Template-DNA, sowie anderen Reaktionssubstanzen genutzt werden. APPLICATION BioThermStarMix is suitable and tested for amplification of genomic targets ranging from l00 bp to 4 kb and of episomal targets (lambda phage; plasmids) up to 10 kb under various reaction conditions. high through-put Hot Start PCR routine diagnostic Hot Start PCR requiring high reproducibility DNA sequencing template preparation STORAGE CONDITIONS BioThermStarMix can be stored at either -20 C in a freezer or at 2-8 C in a usual refrigerator. Shipment at ambient temperature is possible without reduction of Hot Start PCR performance and activity. Storage at 2-8 C is convenient for easy and time-saving assembling of the Hot Start PCR assays. Storage of BioThermStar- Mix at -20 C is recommended for long-term storage after the mix has been used once under nonsterile conditions. Multiple freezing and thawing do not affect the performance or activity of the Mix. At 2-8 C the BioThermStarMix is at least stable for 12 months, in frozen state for 2 years. NOTE Do not contaminate the BioThermStarMix with primers and template DNA used in individual reactions. Thaw and mix all components thoroughly, spin down shortly and chill on ice. It is very important to mix the BioThermStarMix before use to avoid localized concentration! PROTOCOL 1 Place the Hot Start PCR tubes on ice. 2 Prepare first a template/primer mix according to the volumes given in the table below for different reaction volumes. Mix the template/primer mix and chill on ice. 3 Dispense now the corresponding volume of BioThermStarMix (20 µl for a 50 µl reaction) folllowed by the template/primer mix into each reaction tube. Close tubes and mix well. Spin down shortly, if necessary, and chill the tubes on ice. 4 Start the Hot Start program. Transfer the tubes directly from ice into the thermal cycler when the temperature of the block has reached 95 C. Incubate ca. 7 min. at 95 C before cycling. IMPORTANT NOTE The stabilizers in the BioThermStarMix are potential growth substrates for bacterial contaminations! If the Mix has been opened and used under non-sterile conditions, the residual moiety should be used during the next 2 weeks with intermediate storage at 2-8 C or should be frozen at -20 C for longer storage!

21 BioThermStarMix PCR Sterile redi- Sense Antisense Template 2.5x PCR Volume stilled H 2 O Primer Primer DNA Mix 100 µl up to 60 µl x µl y µl z µl 40 µl 50 µl up to 30 µl x µl y µl z µl 20 µl 25 µl up to 15 µl x µl y µl z µl 10 µl 20 µl up to 12 µl x µl y µl z µl 8 µl Final concentrations: 200 nm 200 nm ng 1x 21 Other variable reaction conditions (temperatures, cycling times, concentrations of template, primers, magnesium and polymerase) have to be optimized empirically for each template/primers combination. Most PCR applications work at the standard concentration of 1,5 mm Mg 2+ provided with 1x diluted PCR Mix. Optimal Mg 2+ concentration higher than 1,5 mm can be adjusted using a separate magnesium solution or by increasing stepwise (5 µl increments) the amount of BioThermStarMix added to the reaction assay. This approach can also be used to improve the product yield in amplification of difficult targets on complex template DNA (see table below). Optimization by adding variable amounts of concentrated BioThermStarMix to a 50 µl-assay: Volume of Volume of Final Mg 2+ Final dntp- Taq units Final 2,5x PCR template/ Concen- Concen- per concen- Mix primer mix tration tration reaction trations 20 µl 30 µl 1.5 mm 200 µm 1.25 u 1x 25 µl 25 µl 2.0 mm 250 µm 1.6 u 1.25x 30 µl 20 µl 2.25 mm 300 µm 1.9 u 1.5x GC ml

22 22 BioThermAB DNA POLYMERASE BioThermAB polymerase offers all the benefits of BioTherm DNA polymerase plus an antibody-based, built-in hot start. The BioThermAB polymerase mix contains Hot Start Taq Monoclonal Antibody, which blocks polymerase activity prior to the onset of thermal cycling. This prevents primer-dimers and other artifacts resulting from low-level synthesis from nonspecifically primed sites. The antibodies are quickly inactivated by the increased temperature of thermal cycling. BioThermAB polymerase requires no prolonged heating or denaturing step as do other hot-start methods, making BioThermAB polymerase more convenient and easy to use. BioThermAB weist neben allen Vorteilen einer Bio- Therm DNA-Polymerase die Möglichkeit auf, diese in Hot-Start-PCR-Reaktionen einzusetzen. Zusätzlich enthält dieser BioThermAB Polymerase-Mix Hot- Start-Taq monoklonale Antikörper, welche die Polymerase-Aktivität vor dem Beginn der PCR-Zyklen blokkiert. Dies soll Primer-Dimere und andere Artefakte aufgrund unspezifischer low-level Synthesen verhindern. Die Antikörper werden dann durch die steigende Temperatur der PCR-Zyklen rasch inaktiviert. BioThermAB Polymerase benötigt keine anhaltenden Heiz- oder Denaturierungsphasen wie in anderen Hot-Start Protokollen, was diese Polymerase komfortabler und leichter zu Handhaben macht. APPLICATION Hot-Start-PCR PCR with high specificity CONCENTRATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acid-insoluble form in 30 min at 72 C. STORAGE BUFFER 10 mm K-phosphate buffer ph 7.0, 100 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 0.01% Tween 20; 50% glycerol (v/v) STORAGE TEMPERATURE -20 C 10X REACTION BUFFER 160 mm (NH 4 ) 2 SO 4, 670 mm Tris-HCl ph 8.8 (at 25 C), 15 mm MgCl 2, 0.1% Tween 20 The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. QUALITY CONTROL Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases COMPANION PRODUCTS BioThermStar, MagicTubes GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

23 HOT START TAQ MONOCLONAL ANTIBODY CLONE 8C1 23 The Hot Start Taq Monoclonal Antibodies were derived from a hybridoma (fusion of mouse myeloma cell and the cells after mouse immunization with Taq DNA Polymerase). Hot Start Taq Monoclonal Antibody is mouse IgG 2b isotype. Hot Start Taq Monoclonal Antibody inhibits polymerase activity before the onset of thermal cycling, preventing nonspecific amplification and primer-dimer formation. When the temperature is raised, the antibody is quickly inactivated and PCR proceeds. Hot Start Taq Monoclonal Antibody is effective with a variety of commercially available Taq DNA polymerases (native or recombinant). The use of Hot Start Taq Monoclonal Antibody significantly improves the specificity of PCR amplification what is especially important for PCR-based diagnostics. Die Hot Start Taq Monoklonalen Antikörper erhält man aus einer murinen, hybriden Zelle (Fusion einer murinen Myelom-Zelle und murinen Zelllen nach Immunisierung der Maus mit Taq DNA-Polymerase). Die Antikörper haben den Isotyp IgG 2b. Hot Start Taq Monoklonale Antikörper blockieren die Polymerase-Aktivität vor dem Beginn der PCR-Zyklen, was Primer-Dimere und andere Artefakte aufgrund unspezifischer Amplifikationen verhindert. Die Antikörper werden dann durch die steigende Temperatur der PCR-Zyklen rasch inaktiviert. Die Hot Start Taq Monoklonalen Antikörper funktionieren mit einer Reihe von kommerziell erhältlichen Taq-DNA-Polymerasen (nativ oder rekombinant). Die Verwendung dieser Antikörper verbessert signifikant die Spezifität der DNA-Amplifikation, was speziell in der Diagnostik mit PCR sehr wichtig ist. APPLICATION Hot Start PCR PCR diagnostics CONCENTRATION 5 units/µl UNIT DEFINITION One unit is defined as the amount of Hot Start Taq Monoclonal Antibody required to block 50% of activity of 1 µg of Taq DNA Polymerase at 37 C. STORAGE BUFFER 10 mm Tris-HCl (ph 7.0 at 22 C), 50 mm KCl, 0.1 mm EDTA, 50% glycerol STORAGE TEMPERATURE -20 C REACTION BUFFER The Hot Start Taq Monoclonal Antibody reaction buffer is the same buffer used for the thermostable DNA polymerase. PURITY More than 90% in SDS electrophoresis in 15% PAAG. ASSOCIATE ACTIVITIES No conversion to the covalently closed circular DNA to the nicked or linear form was observed after incubation of 1 µg of puc19 with antibodies in final concentration of 6 u/µl in 20 µl of reaction mixture containing 25 mm Tris-HCl (ph 7.9), 100 mm NaCl, 10 mm MgCl 2 after 16 hours at 37 C. PROTOCOL Add 5 u (1µl) Hot Start Taq Monoclonal Antibody to 100 u enzyme. GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

24 HOT START PCR COMPOUND 24 TYPE HSplus Hot Start (HS) PCR is commonly used to enhance the specificity and sensitivity of PCR amplification. In many cases, HS-PCR has yielded single products in a greater yield than it has been possible with conventional PCR. Usually HS-PCR is inconvenient, time-consuming and incurs a risk of cross contamination. This inconveniences are overcome by HSplus compound. HSplus is used to block Taq polymerase activity during set-up of the PCR reaction at ambient temperatures (20-25 C). The inhibition of Taq polymerase is completely reversed when the temperature is raised above 48 C. HSplus is effective with a variety of commercially available Taq DNA polymerases (native or recombinant). The use of HSplus significantly improves the specificity of PCR amplification what is especially important for PCR-based diagnostics. Hot Start (HS) PCR wird üblicherweise verwendet, um die Spezifität und Sensitivität der PCR-Amplifikation zu steigern. In vielen Fällen erhält man mit der HS-PCR die einzelnen Produkte in einer größeren Ausbeute als es mit einer herkömmlichen PCR möglich ist. Normalerweise ist eine HS-PCR relativ unbequem, zeitintensiv und beinhaltet das Risiko von Kreuz-Kontaminationen. Diese Nachteile kann man bei Verwendung von Hsplus Compound umgehen. HSplus wird verwendet, um die Taq-Polymerase-Aktivität während des Ansetzens der Reaktion bei Umgebungstemperaturen von C zu blockieren. Wenn die Temperatur dann über 48 C steigt, stellt sich die volle Aktivität der Polymerase wieder ein. HSplus funktionieren mit einer Reihe von kommerziell erhältlichen Taq-DNA-Polymerasen (nativ oder rekombinant). Die Verwendung von HSplus verbessert signifikant die Spezifität der DNA-Amplifikation, was speziell in der Diagnostik mit PCR sehr wichtig ist. APPLICATION Hot Start PCR PCR diagnostics CONCENTRATION 1 unit/µl UNIT DEFINITION One unit of activity is the amount of HSplus required for 2 units Taq polymerase per reaction. STORAGE BUFFER 20mM Tris-HCl; ph 8,0; 0,1 mm EDTA STORAGE TEMPERATURE -20 C REACTION BUFFER The HSplus reaction buffer is the same buffer used for the thermostable DNA polymerase. QUALITY CONTROL Activity, PAGE purity, absence of any enzyme contamination. IMPOTRANT NOTE Do not use HSplus when the PCR annealing cycle temperature is below 48 C. PROTOCOL Mix 1 unit of HSplus with 2 units of Taq polymerase in the PCR reaction buffer in presence of all components with the exception of the DNA template investigated. Incubate 10 min at room temperature, then add DNA and start PCR. Add in the same manner HSplus in the positive control reaction. The concentration of HSplus can be 2-10 times decreased in case of absence of its positive action. GC GC GC GC GC u 250 u 500 u 1000 u 5000 u

25 KlenTherm DNA POLYMERASE KlenTherm DNA polymerase is thermostable polymerase corresponding to the KlenTaq polymerase described by W. M. Barnes. It is a N-terminally truncated Taq DNA polymerase. As expressed from a gene construct in E.coli, translation initiates at Met 236, bypasssing the 5'-3' exonuclease domain of the DNA polymerase-encoding gene. This deletion leaves a highly active and even more heat-stable DNA polymerase activity. Repeated exposure to 98ºC, in the recommended reaction buffer, does not seem to diminish the enzyme activity. Significant activity remains even after exposure to 99ºC. The full length enzyme does not tolerate these treatments. Therefore KlenTherm DNA polymerase is an excelllent alternative to modified T7 RNA polymerase in thermal sequencing methods. Even problematic DNA templates with secondary structures and GC-rich regions can be sequenced at 70 C. You can use KlenTherm DNA polymerase also for Long-PCR up to 35 kb in combination with thermostable proof-reading polymerases (e.g. Accu- Therm ). GeneCraft offers several mixtures of Klen- Therm DNA polymerase called Synergy. In special applications KlenTherm DNA polymerase has proven better specificity than regular Taq polymerase. This results in minimising of unspecific DNA amplification products. KlenTherm DNA polymerase is similar to, yet disitinct from, USB Taq and Cetus Stoffel fragment. You will need more KlenTherm than Taq protein if the nucleic acid incorporation is more than 500 bp. Klen- Therm DNA polymerase is shipped at higher (10 u/µl) concentration, so that it can easily incorporate 2 kb, if the same quantity is used as for full-length Taq. The use of KlenTherm is espacially recomended for amplifications of small fragments from genomic DNA. The 10x reaction buffer (on request with or without MgCl 2 ) is delivered free of charge. KlenTherm has a very low 3 -A-Overhang-adding activity. KlenTherm DNA polymerase ist eine thermostabile Polymerase, welche der von W. M. Barnes beschriebenen KlenTaq Polymerase entspricht. Bei der Expression des Genkonstrukts in E.coli beginnt die Translation erst bei Met 236, wodurch die 5-3 Exonucleasedomäne am N-Terminus wegfällt. Diese Deletion führt zu erhöhter Genauigkeit und Aktivität. Ausserdem macht sie die Polymerase hitzestabiler. Wiederholtes Erhitzen auf 98 C im entsprechenden Reaktionspuffer beeinträchtigt nicht die Aktivität! Selbst nach Erhitzung auf 99 C behält KlenTherm ihre Aktivität! Solch eine Hitzebeständigkeit besitzt normale Taq Polymerase nicht. Dadurch ist sie auch für die thermale DNA Sequenzierung eine hervorragende Alternative zu modifizierten T7 DNA Polymerasen. Besonders problematische DNA Templates mit Sekundärstrukturen und hohen GC- Gehalten können mit KlenTherm DNA Polymerase bei 70 C optimal sequenziert werden. Weiterhin ist die KlenTherm DNA Polymerase in Kombination mit einer thermostabilen proofreading -DNA Polymerase (AccuTherm ) für die Amplifikation von besonders langen DNA Fragmenten bis 35 kb geeignet. GeneCraft bietet solche Mischungen unter dem Namen Synergy an. In bestimmten Applikationen kann KlenTherm DNA Polymerase eine optimalere Spezifität als herkömmliche Taq Polymerasen aufweisen, was zur Minimierung von unspezifischen DNA-Amplifikationsprodukten beitragen kann. Gerade bei kurzen Fragmenten genomischer DNA ist KlenTherm DNA Polymerase sehr zu empfehlen. KlenTherm DNA polymerase ist vergleichbar mit Taq (USB) und Stoffel fragment (Cetus). Wir empfehlen, bei Fragmenten über 500 bp mehr Units von KlenTherm DNA Polymerase einzusetzen als bei gewöhnlicher Taq Polymerase. Aus diesem Grund liefern wir Ihnen KlenTherm DNA polymerase in einer Konzentration von 10 u/µl. KlenTherm DNA Polymerase wird mit einem 10x Reaktionspuffer (auf Wunsch auch mit MgCl 2 als separater Lösung) ohne Aufpreis geliefert. KlenTherm zeigt eine sehr niedrige Aktivität 3 -A-Überhänge anzuhängen. 25

26 KlenTherm DNA POLYMERASE 26 APPLICATIONS Fidelity The relative mutation rate during polymerization is twofold lower for KlenTherm as compared to the fulllength Taq DNA polymerase. Cycle sequencing The absence of the 5'-3' exonuclease activity makes KlenTerm especially suitable for cycle sequencing. It gives higher sequence intensity and very low backgrounds. Long PCR KlenTherm in combination with a Pfu DNA polymerase (AccuTherm ) exhibiting a proof-reading activity can amplify up to 35 kb DNA fragments. Mutation analysis KlenTherm has a reduced tendency to extend a mismatched 3'-oligonucleotide end making it suitable for mutation analysis with mutationspecific oligos (ARMS analysis). CONCENTRATION 10 units/µl UNIT DEFINITION One unit is defined as the amount of enzyme that incorporates 10 nmoles of dntps into acidinsoluble form in 30 min at 72ºC under the assay conditions 25 mm TAPS (tris-(hydroxy-methyl)-methyl-amino-propanesulfonic acid, sodium salt) ph 9.3 (at 25ºC), 50 mm KCl, 2 mm MgCl 2, 1 mm ß-mercaptoethanol) and activated calf thymus DNA as substrate. STORAGE BUFFER 10 mm K-phosphate buffer ph 7,0; 100 mm NaCl; 0,5 mm EDTA; 1 mm DTT; 0,01% Tween 20; 50% glycerol (v/v) STORAGE TEMPERATURE Store KlenTherm DNA polymerase below 0 C, preferably at -20 C, in a constant temperature freezer. 10X REACTION BUFFER 500 mm KCl, 100 mm Tris-HCl (ph 9 at 25 C), 1% Triton X100, 35 mm MgCl ml 10x reaction buffer Cat. No GC Please note the difference between KlenTherm and BioTherm reaction buffers! COMPANION PRODUCTS KlenThermN DNA polymerase, KlenThermase, KlenThermaseN, Synergy DNA polymerase, SynergyN DNA polymerase, SynergyT DNA polymerase, dntps AMPLIFICATION CONDITIONS 10x reaction buffer 3 µl 50 mm MgCl µl dntp Mix10 (end concentration 200 µm) 0.6 µl human genomic DNA ( ng) 1 µl forward primer (25 pm) 2 µl reverse primer (25 pm) 2 µl KlenTherm (10 units/µl) 0.5 µl H 2 O 18.8 µl 58ºC 0.5 min 72ºC 4 min 93ºC 20 sec 30 cycles total 30 µl Fragment is about 1,5 kb GC GC GC GC GC u 250 u 500 u 1000 u 5000 u