| SKU | Size | Availability | Price | Qty |
|---|---|---|---|---|
| B744267-4KU | 4KU | Available within 8-12 weeks(?) Production requires sourcing of materials. We appreciate your patience and understanding. | $283.90 |
| Specifications & Purity | EnzymoPure™, Free of DNA endonuclease and exonuclease. |
|---|---|
| Stability And Storage | Store at -20℃, stable for at least 1 year. |
| Storage Temp | Store at -20°C |
| Shipped In | Ice chest + Ice pads |
| Grade | EnzymoPure™ |
| Product Description | Aladdin's Bst DNA Polymerase, Large Fragment is the large fragment of Bacillus stearothermophilus (Bst) DNA Polymerase I with 5'→3' DNA polymerase activity, but not 3'→5' and 5'→3' exonuclease activities. Bst DNA Polymerase, Large Fragment has strong strand displacement ability and can be applied to isothermal nucleic acid amplification reactions, such as loop-mediated isothermal amplification (LAMP) and Rolling-circle amplification (RCA), etc.The temperature of isothermal amplification mediated by Bst DNA Polymerase, Large Fragment is generally 50-68℃, usually 65℃. Compared with Bst DNA Polymerase 2.0, this product does not have the ability to incorporate dUTP into newly synthesized DNA during isothermal amplification.Bst DNA Polymerase has 5'→3' exonuclease activity, while Bst DNA Polymerase, Large Fragment lacks 5'→3' exonuclease activity by deletion mutation.Please refer to Figure 1 for the performance of Aladdin's Bst DNA Polymerase, Large Fragment. Figure 1. The performance of Aladdin's Bst DNA Polymerase, Large Fragment in LAMP. Reactions of 25µl in a final volume containing 8U of this product or a similar product from Competitor and the indicated amounts of CaMV35 promoter of tobacco mosaic virus were incubated at 65℃ for 1h, inactivated at 80 ℃ for 20min, and then examined by 1.5% agarose gel electrophoresis. As shown in the figure, compared with the competitor, this product has a slightly better or at least equivalent performance. M, DNA Ladder . Application： Loop-mediated isothermal amplification (LAMP), helicase isothermal gene amplification (HDA) and other DNA isothermal amplification; multiple displacement amplification (MDA); whole genome amplification (WGA); high GC content DNA sequencing; rapid sequencing of nanogram-level DNA templates; library preparation for DNA sequencing, etc.Unit definition: One unit is defined as the amount of enzyme that incorporates 10nmol of dNTPs into acid insoluble material in 30 minutes at 65℃.This product is in lyophilized form and needs to be reconstituted before use. Recommended reconstitution buffer: 10mM Tris-HCl (pH 7.5), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol.10X Bst Reaction Buffer: 200mM Tris-HCl, 100mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton X-100, pH 8.8 at 25°C.Inactivation or inhibition: Heat at 80℃ for 20 minutes.Source： The recombinant large fragment of Bacillus stearothermophilus DNA Polymerase I expressed in E. coli.Purity: Free of DNA endonuclease and exonuclease.Applications: Loop-mediated isothermal amplification (LAMP), helicase isothermal gene amplification (HDA) and other DNA isothermal amplification; multiple displacement amplification (MDA); whole genome amplification (WGA); high GC content DNA sequencing; rapid sequencing of nanogram-level DNA templates; library preparation for DNA sequencing, etc.Inactivation or inhibition： Heat at 80℃ for 20 minutes.Precautions： This product is a lyophilized powder and needs to be reconstituted before use. Please use the recommended buffer for reconstitution. After reconstitution, the enzyme should be aliquoted and stored at -20°C to avoid repeated freeze-thaw.The temperature for isothermal amplification should not be higher than 70℃. Otherwise, the enzyme will be inactivated.Bst DNA Polymerase, Large Fragment cannot be used for thermal cycle sequencing and PCR.Always set up a negative control without template DNA for each isothermal amplification experiment to exclude background amplification.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use： 1.Primer Design:For the design of loop-mediated isothermal amplification primers, please refer to http://primerexplorer.jp/e/ and version V5 is recommended. The manual can be downloaded at http://primerexplorer.jp/e/v5_manual/index.html. Refer to this manual for the preliminary screening of loop-mediated isothermal amplification primers (LAMP), and more suitable primers need to be verified by experiments. LAMP primers consist of 4 or 6 (including Loop) primers. It is recommended to design 6 primers for the experiment2.Set up LAMP reactions on ice as follows: Reagent Volume Finalconcentration Nuclease-Free Water (13.5-x)μl - 10X BstReaction Buffer 2.5μl 1X MgSO4 (100mM) 1.5μl 6mM (8mMtotal) dNTP (10mM each) 3.5μl 1.4mM each FIP/BIP Primers (25X,40μM) 1μl 1.6μM F3/B3Primers (25X,5μM) 1μl 0.2μM LoopF/BPrimers (25X,10μM) 1μl 0.4μM Template xμl ＞ 10 copies or more BstDNA Polymerase, Large Fragment (8U/μl) 1μl 320U/ml Totalvolume 25μl - Note 1: After preparing the reaction, add 1μl of high-concentration SYBR Green I to each tube. After isothermal amplification, centrifuge at 8,000g for 1 min, and the positive reaction turns fluorescent green, while the negative reaction remains colorless or brown. Even without any indicators, the positive reactions can be identified by the turbidity of reactions. Negative reactions are clear and transparent.Note 2: To optimize the reaction, adjust the Mg2+ concentration (4-10mM), the amount of enzyme (0.04-0.32U/μl) or change the reaction temperature (50-68℃).Note 3: If analysis is performed by agarose gel electrophoresis or other methods that require opening of the LAMP reaction vessel, set up auxiliary analysis areas and equipment to avoid contamination.Note 4: In order to ensure the reproducibility of the experiment, it is recommended to add template DNA at the last.Note 5: It is strongly recommended to set a negative control without template to confirm the specificity of amplifications.Note 6: To prevent contamination, the reaction mixture should be prepared on a clean bench.Note 7: The preparation of reagents and template DNA should be performed in different areas from the electrophoresis area to avoid contamination.3.Incubate at 65℃ for 60min.4.Incubate at 80℃ for 20min to terminate the reaction.5.If necessary, examine the reaction product by 1.5% agarose gel electrophoresis. The amplification products should show a ladder-like band pattern.Related Products:Cat. No.Product NamePack Size D7050S BstDNA Polymerase, Large Fragment 800U D7050M BstDNA Polymerase, Large Fragment 4000U D7049M BstDNA Polymerase, Large Fragment(powder) 4000U D7053S phi29 DNA Polymerase 250U D7053M phi29 DNA Polymerase 1kU D7053L phi29 DNA Polymerase 5kU D7053XL phi29 DNA Polymerase 20kU D7055S BsuDNA Polymerase, Large Fragment 200U D7055M BsuDNA Polymerase, Large Fragment 1000U D7359-250µl dUTP (100mM) 250µl D7359-1ml dUTP (100mM) 1ml D7360S Uracil-DNA Glycosylase (E. coli) 1000U D7360M Uracil-DNA Glycosylase (E. coli) 5000U D7362S Uracil-DNA Glycosylase (Heat-labile, Bacterium) 100U D7362M Uracil-DNA Glycosylase (Heat-labile, Bacterium) 500U D7364S Uracil-DNA Glycosylase (Heat-labile, Cod) 200U D7364M Uracil-DNA Glycosylase (Heat-labile, Cod) 1000U D7364L Uracil-DNA Glycosylase (Heat-labile, Cod) 5000U D7371 dNTP Mixture (2.5mM each) 1ml D7373 dNTP Mixture (25mM each) 250μl D7376-1ml dNTP/dUTP Mixture (2.5mM each/5mM) 1ml D7376-5ml dNTP/dUTP Mixture (2.5mM each/5mM) 5ml |
