| Description | Aladdin's E. coli DNA Polymerase I purified using the PerfectProtein™ technology platform developed by aladdin, catalyzes the the 5'→3' polymerization of deoxyribonucleotides in a DNA template-dependent manner [1]. It also possesses dsDNA nick-specific 5'→3' exonuclease activity, Aladdin's E. coli DNA Polymerase I purified using the PerfectProtein™ technology platform developed by aladdin, catalyzes the the 5'→3' polymerization of deoxyribonucleotides in a DNA template-dependent manner [1]. It also possesses dsDNA nick-specific 5'→3' exonuclease activity, ssDNA-specific 3'→5' exonuclease activity, and RNase H activity [2]. The DNA synthesis activity and dsDNA nick-specific 5'→3' exonuclease activity allow for DNA synthesis starting from a 3'-OH group at the nick and degradation of single-stranded DNA at 5' end, facilitating gap filling. Its ssDNA-specific 3'→5' exonuclease activity serves a proofreading function during DNA synthesis. In the presence of dNTP, the E. coli DNA Polymerase I primarily exhibits DNA polymerase activity, while in the absence of dNTP, it displays more ssDNA-specific exonuclease activity, such as 5'→3' exonuclease activity on either strand at the 5' end of a blunt-ended dsDNA.The versatility of E. coli DNA Polymerase I allows it to initiate the synthesis of new DNA chains from gaps or nicks in dsDNA, and to degrade the DNA strand complementary to the template strand from the nick, enabling nick translation. It also ensures proofreading of mismatch during DNA replication and fills in gaps that occur during replication and repair processes [3].Please refer to Figure 1 for the performance of this product in filling 5' overhangs of dsDNA.Figure 1. Performance of Aladdin's E. coli DNA Polymerase I in filling 5' overhangs of dsDNA. In a 20µl reaction (10mM Tris-HCl, 50mM NaCl, 10mM MgCl2, 1mM DTT, 100µM dNTP Mix, 0.5µM dsDNA with 5' overhang, pH 7.9 at 25℃), the specified amount of this product or E. coli DNA Polymerase I from Company N (Competitor) was added. After incubation at 37℃ for 20 minutes, reactions were terminated by incubation at 75℃ for 20 minutes, followed by 15% native PAGE analysis of 5µl of the reaction product after mixing with 1µl of 6X DNA Loading Buffer. Electrophoresis was conducted at 180V for 60 minutes, and then the gel was stained with Gel-Red (10000X) at room temperature for 5 minutes. The experimental results were observed under a UV lamp. As shown in the figure, this product has similar catalytic efficiency to Competitor. The substrate dsDNA with 5' overhang was obtained by annealing 5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3' and 5'-GTAAAACGACGGCCAGT-3' using Annealing Buffer for DNA Oligos (5X) according to manufacture's instructions. This figure is for reference only, which may vary due to different experimental conditions.Please refer to Figure 2 for performance of this product in digesting double-stranded linear DNA with amino-modified 3' ends.Figure 2. Performance of Aladdin's E. coli DNA Polymerase I in digesting amino-modified 3' ends of dsDNA (5'→3' exonuclease activity). In a 20µl reaction (10mM Tris-HCl, 50mM NaCl, 10 mM MgCl2, 1mM DTT, 0.5µM dsDNA), the specified amount of this product or E. coli DNA Polymerase I from Company N (Competitor) was added. After incubation at 37℃ for 20 minutes, reactions were terminated by incubation at 75℃ for 20 minutes, followed by 15% native PAGE analysis of 5µl of the reaction product after mixing with 1µl of 6X DNA Loading Buffer. Electrophoresis was conducted at 180V for 60 minutes, and then the gel was stained with Gel-Red (10000X) at room temperature for 5 minutes. The experimental results were observed under a UV lamp. As shown in the figure, this product has similar catalytic efficiency to Competitor. The substrate dsDNA with amino-modified 3' ends was obtained by annealing 5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3'NH2 and 5'-GTAAAACGACGGCCAGTCTATGTATCTATGTAT-3'NH2 using Annealing Buffer for DNA Oligos (5X) according to manufacture's instructions. This figure is for reference only, which may vary due to different experimental conditions.sApplication：DNA synthesis; complementary filling of dsDNA 5' overhangs; removal of dsDNA 3' overhangs; second strand cDNA synthesis [4]; in combination with DNase I for DNA nick translation; nick translation to obtain probes with high specific activity.Source：Purified from E. coli with recombinant expression of E. coli DNA Polymerase I.Enzyme storage buffer：25mM Tris-HCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH 7.4 at 25 ℃).Inactivation or inhibition：This product can be inactivated by incubation at 75℃ for 20 minutes.Precautions：Due to the exonuclease activity of E. coli DNA Polymerase I, please avoid high environmental temperatures before performing the reaction. Otherwise, the DNA strands may be cleaved.E. coli DNA Polymerase I does not possess endonuclease activity, nor DNase I either. Therefore, when performing nick translation reactions, DNase I must be added.Vigorous shaking or stirring of E. coli DNA Polymerase I can cause enzyme inactivation.E. coli DNA Polymerase I has a high affinity for DNA. Addition of excessive amount of enzyme may lead to aggregation, thus affecting the amplification reactions.E. coli DNA Polymerase I can polymerize deoxyribonucleotides labeled with biotin, digoxigenin, or fluorescence, etc, allowing for synthesis of labeled DNA probes.The enzyme should be kept on ice during use, and stored at -20℃ immediately after use.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. Fill-in of 5' overhangs of dsDNAa. Set up the following reaction on ice.ReagentVolumeFinal ConcentrationNuclease-free Water(16-x)µl-dsDNA with 5' overhangsxµl~0.5µM or 5-200ng/µl10X Reaction Buffer2µl1XdNTP Mix (2mM each)1µl100µME.coli DNA Polymerase I (10U/µl)1µl0.5U/µlTotal Volume20µl-Note 1: The enzyme amount can be reduced appropriately to avoid template cleavage due to its exonuclease activity.Note 2: When multiple reactions are required, prepare a master mix including all reagents except for dsDNA, and then dispense to different nuclease-free PCR tubes. Finally, add dsDNA template to each tube.Note 3: If the dsDNA with 5' overhangs are oligonucleotides, the final concentration can be approximately 0.5µM. However, for digested DNA plasmids, the final concentration can be approximately 5-200ng/µl.b. Mix well gently and then have a pulse-spin in a microfuge to collect the liquid at the bottom of the tube.c. Incubate at 37℃ for 20 minutes. Note: The reaction time can be adjusted based on actual situations.d. Incubate at 75℃ for 20 minutes to inactivate the E. coli DNA Polymerase I.2. Digestion of Double-stranded Linear DNAa. Set up the following reaction on ice.ReagentVolumeFinal ConcentrationNuclease-free Water(17-x)µl-dsDNAxµl~0.5µM or 5-200ng/µl10X Reaction Buffer 2µl1XE.coli DNA Polymerase I (10U/µl)1µl0.5U/µlTotal Volume20µl-Note: When multiple reactions are required, prepare a master mix including all reagents except for dsDNA, and then dispense to different nuclease-free PCR tubes. Finally, add dsDNA to each reaction tube.b. Mix well gently and then have a pulse-spin in a microfuge to collect the liquid at the bottom of the tube.c. Incubate at 37℃ for 20 minutes. Note: The reaction time can be adjusted based on actual situations.d. Incubate at 75℃ for 20 minutes to inactivate E. coli DNA Polymerase I.3. For other applications, please refer to appropriate literature.FAQ:1. Can the E. coli DNA Polymerase I fill in 3' overhangs?No, E. coli DNA Polymerase I cannot fill in 3' overhangs. It can only generate blunt ends by removing 3' overhangs. 's E. coli DNA Polymerase I, Klenow Fragment, and T4 DNA Polymerase can be used for fill-in of 3' overhangs.2. Can E. coli DNA Polymerase I fill in 5' overhangs of DNA?Yes, E. coli DNA Polymerase I can fill in 5' overhangs of dsDNA. Klenow Fragment (, D7037) lacks 5'→3' exonuclease activity and is recommended for fill-in of 5' overhangs.3. Can E. coli DNA Polymerase I be used for nick translation experiments?Yes, nick translation experiments are one of the important applications of E. coli DNA Polymerase I.4. Are there temperature requirements for nick translation experiments?The incubation temperature for nick translation experiments should be below 20℃. At higher temperatures, the newly synthesized DNA can separate and be replicated.5. Can E. coli DNA Polymerase I be heat-inactivated?Yes, this product can be inactivated by heating at 75℃ for 20 minutes. Addition of 10mM EDTA to chelate Mg2+ before performing heat-inactivation can protect the DNA ends. 6. Can E. coli DNA Polymerase I remove 5' overhangs?No, the 5'→3' exonuclease activity of this product is only applicable to gaps in dsDNA.7. Can DNA nick translation be used for labeling probes?Yes, this product can remove template bases at nicks using its 5'→3' exonuclease activity and fill in nicks with labeled nucleotides. This method is suitable for generating large and uniform probes, but with lower efficiency probably.References:1. Kunkel TA, Loeb LA, Goodman MF. J Biol Chem. 1984. 259(3):1539-45.2. Green MR, Sambrook J. Cold Spring Harb Protoc. 2020. 2020(5):100743.3. Yu H, Chao J, Patek D, Mujumdar R, Mujumdar S, Waggoner AS. Nucleic Acids Res. 1994. 22(15):3226-32.4. D'Alessio JM, Gerard GF.Nucleic Acids Res. 1988. 16(5):1999-2014... Read More | p53 and MDM2 proteins-interaction-inhibitor (chiral) (Compound 32) is an inhibitor of the interaction between p53 and MDM2 proteins | Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed polymerase activity. In addition reverse transcriptases catalyze the degradation of RNA in an RNA-DNA hybrid. The exonucleolytic activity proceeds in a 5' ---> 3' direction. The RNA or DNA directed activity requires a template (RNA or DNA) and a primer. The following is a schematic illustration of the reaction:Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) 12-18 as the template-primer in 20 minutes at 37° C.ApplicationsHIV reverse transcriptase is used for research on the AIDS primer. However it can be substituted for AMV reverse transcriptase, which is mainly used to transcribe mRNA into double stranded cDNA, that can be inserted into prokaryotic vectors. The enzyme can also be used with either single stranded DNA or RNA templates to make probes for use in hybridization experiments. It can be used for labeling the termini of DNA fragments with protruding 5' termini. The enzyme can also be used to sequence DNAs by the dideoxy chain termination method of Sanger when the Klenow fragment of E. coli DNA polymerase I, or the T7 DNA polymerase yield unsatisfactory results.Reagents0.05 M Tris, pH 8.3, containing 0.008 M MgCl21 mg/ml polyadenylic acid in water (poly A)DNA primer:Oligo d(T)12-181 µ mole dTTP/mL stock solution[methyl-3H]-Thymidine 5'-triphosphate (3H-dTTP)dTTP-3H-dTTP working mix: Add 1-2 µL 3H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 105 cpm/mL1% bovine serum albumin10% perchloric acid1% perchloric acidBuffer substrate reaction mixture: Prepare fresh, immediately before use:For each 1mL of reaction mixture required mix:0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl20.3 mL 1 mg/mL poly(A) RNA template0.005 mL 0.02 mg/mL oligo d(T)12-18 DNA primer0.02mL 1% BSAEnzymedilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl2 containing 0.1 mg/mL (1%) BSAProcedurePipette into each tube as follows:Buffer substrate mix：0.1 mLdTTP-3H3-dTTP：0.1 mLEnzyme：5-10 µLIncubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2µ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.Calculation... Read More | Purity>95% SDS-PAGEFunctionLipid transport protein in adipocytes. Binds both long chain fatty acids and retinoic acid. Delivers long-chain fatty acids and retinoic acid to their cognate receptors in the nucleus | Purity> 97 % by SDS-PAGE and HPLC analyses.FunctionReceptor for TNFSF2/TNF-alpha and homotrimeric TNFSF1/lymphotoxin-alpha. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation whichPurity> 97 % by SDS-PAGE and HPLC analyses.FunctionReceptor for TNFSF2/TNF-alpha and homotrimeric TNFSF1/lymphotoxin-alpha. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis. Contributes to the induction of non-cytocidal TNF effects including anti-viral state and activation of the acid sphingomyelinase... Read More |