| Product 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, 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.s
Application： 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.
|
|---|
