E.coli DNA Polymerase I

大肠杆菌DNA聚合酶I

E.coli DNA Polymerase I

E745501-5000U

5000U

EnzymoPure™

不含除E.coli DNA Polymerase I之外的其它种类的DNA外切酶，不含内切酶，不含RNase。

-20°C储存

超低温冰袋运输

-20ºC保存，两年有效。

阿拉丁生产的E.coli'DNA Polymerase I，即大肠杆菌DNA聚合酶I，是由阿拉丁自主研发的PerfectProtein™技术平台表达、纯化获得的一种DNA模板依赖的DNA聚合酶，可以在单链DNA模板和与其配对的引物存在的条件下，催化5'→3'方向的DNA合成1]。"E.coli'DNA Polymerase I同时还具备双链DNA缺刻(nick)处特异性的5'→3'外切酶活性、单链特异性的3'→5'外切酶活性以及核糖核酸酶H活性2]。"E.coli'DNA Polymerase I的DNA合成酶活性和双链DNA缺刻(nick)处特异性的5'→3'外切酶活性，可以实现缺刻处3'-OH起始的DNA合成和5'端单链DNA的降解，实现缺口平移。"E.coli'DNA Polymerase I的单链特异性的3'→5'外切酶活性可以起到DNA合成的校验作用(proofreading)。在有dNTP存在的情况下，"E.coli DNA Polymerase I更多地表现为DNA聚合酶活性；而当dNTP不存在的情况下，E.coli'DNA Polymerase I更多表现为单链特异性的外切酶活性，例如可以表现为平末端双链DNA中的任一条链的5'端的5'→3'外切酶活性。E.coli DNA Polymerase I的多功能性可实现以双链DNA中的缺刻或缺口为起点合成新的DNA链；从缺口处降解与模板链互补的DNA链，实现DNA的缺口平移；保证DNA复制过程中错配的校对，并对复制和修复中出现的空隙进行填补3]。阿拉丁生产的E.coli'DNA Polymerase I补平双链DNA 5'突出末端(5' overhang)的效果参考图1。图1. 阿拉丁生产的E.coli DNA Polymerase I 补平双链DNA 5'突出末端(5' overhang)的效果图。在20µl反应体系中加入图中指定量的本产品或N公司(Competitor)的"E.coli DNA Polymerase I，37ºC孵育20分钟进行反应，反应完成后75ºC孵育20分钟以终止反应。取出5μl反应产物，加入1μl 6X DNA Loading Buffer ，然后进行15%非变性聚丙烯酰胺凝胶电泳。180V电泳60分钟，之后用Gel-Red (EB升级换代产品，10000X) 室温染色5分钟，在紫外灯下观察实验结果。如图所示，本产品与N公司的产品相比，具有类似的催化效果。反应体系(20µl): 10mM Tris-HCl, 50mM NaCl, 10mM MgCl2, 1mM DTT, 100µM dNTP Mix, 0.5µM dsDNA with 5' overhang (pH7.9 @ 25ºC)。dsDNA with 5' overhang (也称具有5'突出末端的双链线性DNA)是使用Annealing Buffer for DNA Oligos (5X) ("D0251', ")并按照该产品说明书推荐的程序，把5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3'和5'-GTAAAACGACGGCCAGT-3'这两条寡核苷酸链进行退火反应得到的产物，并以此退火产物为底物，进行双链DNA 5'突出末端的补平。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。阿拉丁生产的E.coli'DNA Polymerase I对3'端带有氨基修饰的双链线性DNA消化(5'→3'外切酶活性)的效果参考图2。图2. 阿拉丁生产的E.coli DNA Polymerase I 对3'端带有氨基修饰的双链线性DNA消化(5'→3'外切酶活性)的效果图。在20µl反应体系中加入图中指定量的本产品或N公司(Competitor)的"E.coli DNA Polymerase I，37ºC孵育20分钟进行反应，反应完成后75ºC孵育20分钟以终止反应。取出5μl反应产物，加入1μl 6X DNA Loading Buffer ，然后进行15%非变性聚丙烯酰胺凝胶电泳。180V电泳60分钟，之后用Gel-Red (EB升级换代产品，10000X) 室温染色5分钟，在紫外灯下观察实验结果。如图所示，本产品与N公司的产品相比，具有类似的催化效果。反应体系(20μl)：10mM Tris-HCl, 50mM NaCl, 10mM MgCl2, 1mM DTT, 0.5µM dsDNA。3'端带有氨基修饰的单链DNA不会被"E.coli'DNA Polymerase I 的3'→5'外切酶活性所降解。3'端带有氨基修饰的dsDNA是使用Annealing Buffer for DNA Oligos (5X) ("D0251', ")并按照该产品说明书推荐的程序，把5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3'NH2和5'-GTAAAACGACGGCCAGTCTATGTATCTATGTAT-3'NH2这两条寡核苷酸链进行退火反应得到的产物，并以此退火产物为底物，进行双链线性DNA消化。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。 用途 ： DNA合成；双链DNA 5'突出末端(5' overhang)的补平；双链DNA 3'突出末端(3' overhang)的削平；cDNA第二条链合成[4]；与DNase I一起使用，进行DNA切口平移；DNA的切口翻译以获得具有高比活性的探针。 来源 ： 纯化自携带编码E.coli DNA Polymerase I基因的E.coli重组菌株。 酶储存溶液 ： 25mM Tris-HCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH7.4 @25ºC)。10X Reaction Buffer: 100mM Tris-HCl, 500mM NaCl, 100mM MgCl2, 10mM DTT (pH7.9 @25ºC)。 失活或抑制 ： 75ºC孵育20分钟可使E.coli DNA Polymerase I失活。 注意事项 ： 由于E.coli DNA Polymerase I具有外切酶活性，在进行反应前应尽量避免环境温度偏高导致的DNA链被切除。E.coli DNA Polymerase I不具有内切酶活性，且本产品不包含DNase I，进行切口翻译反应时必须另外添加DNase I。对E.coli DNA Polymerase I剧烈震荡或搅拌会使酶失活。E.coli DNA Polymerase I与DNA的亲和力较高，加入过量的酶易发生凝集作用，影响反应的进行。E.coli DNA Polymerase I可使用带修饰的核苷酸(例如生物素、地高辛、荧光标记核苷酸)作为DNA合成的底物，以用于DNA探针的标记。酶使用时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于-20℃保存。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.双链DNA 5'突出末端(5' overhang)的补平：a.对于双链DNA 5'突出末端(5' overhang)的补平，参考下表在冰浴中配制反应体系(以20μl体系为例)。 Reagent Volume Final Concentration Nuclease-Free Water (16-x)μl - dsDNA with 5' overhang xμl ~0.5µM or 5-200ng/µl 10X Reaction Buffer 2μl 1X dNTP Mix (2mM each) 1μl 100μM E.coli DNA Polymerase I (10U/µl) 1μl 0.5U/µl Total Volume 20μl - 注1：在进行末端补平实验时可适当减少酶量，避免在外切酶活性的作用下导致模板的缺失。注2：如果同时进行多个反应，可以把上表中除dsDNA with 5' overhang之外的所有溶液和酶提前混合，分装到各反应管，最后再加入dsDNA with 5' overhang。注3：dsDNA with 5' overhang如果是寡核苷酸，最终浓度可以约为0.5µM；如果是消化后的DNA质粒等最终浓度可以约为5-200ng/μl。b.按上表设置好反应体系后，适当轻轻混匀反应体系，随后低速离心以使粘附在管壁上的液体沉淀至管底。c.反应条件：37ºC孵育20分钟。注：反应时间可以根据实际情况酌情适当调节。d.终止反应：75ºC孵育20分钟使E.coli DNA Polymerase I失去活性。2.双链线性DNA的消化：a. 对于双链线性DNA消化，参考下表在冰浴中配制如下反应体系(以20μl体系为例)。 Reagent Volume Final Concentration Nuclease-Free Water (17-x)μl - dsDNA xμl ~0.5µM or 5-200ng/µl 10X Reaction Buffer 2μl 1X E.coli DNA Polymerase I (10U/µl) 1μl 0.5U/µl Total Volume 20μl - 注：如果同时进行多个反应，可以把上表中除dsDNA之外的所有溶液和酶提前混合，分装到各反应管，最后再加入dsDNA。b. 按上表设置好反应体系后，适当轻轻混匀反应体系，随后低速离心以使粘附在管壁上的液体沉淀至管底。c. 反应条件：37ºC孵育20分钟。注：反应时间可以根据实际情况酌情适当调节。d. 终止反应：75ºC孵育20分钟使E.coli DNA Polymerase I失去活性。3.其它用途可以参考适当的文献资料进行。常见问题：1.E.coli DNA Polymerase I能补平3'突出末端吗？不能，E.coli DNA Polymerase I只能通过移除3'突出末端的方式形成平末端。E.coli DNA Polymerase I 、Klenow Fragment 、T4 DNA Polymerase是削平3'突出末端的最佳选择。2.E.coli DNA Polymerase I能补平DNA的5'突出末端吗？可以，5'突出末端会被补平，3'突出末端会被削平。Klenow Fragment 缺乏5'→3'外切酶活性，是补平DNA的5'突出末端的首选酶。3.E.coli DNA Polymerase I能用于切口平移实验吗？能，切口平移实验是该酶的重要应用之一。4.切口平移实验时有温度要求吗？切口平移时的孵育温度应低于20ºC。在较高的温度下，新合成的链可以分离并被复制。5.E.coli DNA Polymerase I可以热失活吗？可以。反应结束后添加10mM EDTA螯合Mg2+，当温度升高时，可以保护DNA末端，然后在75ºC加热20分钟即可将酶灭活。6.E.coli DNA Polymerase I能移除5'突出末端吗？ 不能，E.coli DNA Polymerase I的5'→3'外切酶活性仅适用于双链DNA缺刻处。7.DNA切口平移能够用来做标记探针吗？可以，DNA聚合酶I用5'→3'核酸外切酶去除缺口处的前导碱基，并用标记的碱基填充。该方法适于制作大而均匀的探针，但比活性不高。参考文献：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. 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.