| Product Description | Aladdin's Lambda Exonuclease (λ Exonuclease) is a highly processive 5´→3´DNA exonuclease that selectively digests double-stranded DNA (dsDNA) phosphorylated at the 5´end. It also degrades single-stranded DNA (ssDNA) or non-phosphorylated dsDNA, but at greatly reduced rates, and cannot initiate digestion at gaps in DNA [1, 2]. This product cleaves the 5´-OH end approximately 20-fold slower than the 5´-P end, and cleaves ssDNA approximately 100-fold slower than dsDNA.This product is a recombinant enzyme expressed and purified using the PerfectProtein™ Platform developed by aladdin.Please refer to Figure 1 for the performance of Aladdin's Lambda Exonuclease in digesting linear dsDNA with 5´ phosphorylation on one strand.Figure 1. Digestion of linear dsDNA with 5´ phosphorylation on one strand by Aladdin's Lambda Exonuclease . In 20µl reactions containing 67mM Glycine-KOH pH9.4, 2.5mM MgCl2, 50µg/ml BSA and 5pmol of 26bp linear dsDNA, the indicated amounts of Lambda Exonuclease from aladdin or Competitor were added. The reactions were incubated at 37℃ for 30 minutes and then terminated by the addition of 10 mM EDTA followed by incubation at 75℃ for 10 min. The reaction products were run on 10% native PAGE at 180V for 40 minutes with TBE electrophoresis buffer. The gel was stained with NA-Red at room temperature for 7 minutes before taking photos for observation. As shown in the figure, this product has comparable performance to the Lambda Exonuclease from Competitor N. This figure is for reference only.Please refer to Figure 2 for the performance of Aladdin's Lambda Exonuclease in digesting linear ssDNA with 5´ phosphorylation.Figure 2. Digestion of linear ssDNA with 5' phosphorylation by Aladdin's Lambda Exonuclease . In 20µl reactions containing 67mM Glycine-KOH pH9.4, 2.5mM MgCl2, 50µg/ml BSA and 10pmol of 26nt linear ssDNA, the indicated amounts of Lambda Exonuclease from aladdin or Competitor were added. The reactions were incubated at 37℃ for 30 minutes and then terminated by the addition of 10 mM EDTA followed by incubation at 75℃ for 10 min. The reaction products were run on 10% native PAGE at 180V for 40 minutes with TBE electrophoresis buffer. The gel was stained with NA-Red at room temperature for 7 minutes before taking photos for observation. As shown in the figure, this product has comparable performance to the Lambda Exonuclease from Competitor N. This figure is for reference only.Please refer to Figure 3 for the performance of Aladdin's Lambda Exonuclease in digesting linear dsDNA without 5´ phosphorylation. Figure 3. Digestion of linear dsDNA without 5´ phosphorylation by Aladdin's Lambda Exonuclease . In 20µl reactions containing 67mM Glycine-KOH pH9.4, 2.5mM MgCl2, 50µg/ml BSA and 5pmol of 26bp linear dsDNA without 5' phosphorylation, the indicated amounts of Lambda Exonuclease from aladdin or Competitor were added. The reactions were incubated at 37℃ for 30 minutes and then terminated by the addition of 10 mM EDTA followed by incubation at 75℃ for 10 min. The reaction products were run on 10% native PAGE at 180V for 40 minutes with TBE electrophoresis buffer. The gel was stained with NA-Red at room temperature for 7 minutes before taking photos for observation. As shown in the figure, this product has comparable performance to the Lambda Exonuclease from Competitor N. This figure is for reference only.s
Application： Generation of single-stranded PCR products for DNA sequencing; DNA single-stranded conformational polymorphism (SSCP) analysis; rolling cycle amplification; generation of ssDNA from dsDNA; cloning of PCR products; plasmid preparation and purification.
Source： Purified from a E. coli strain expressing the recombinant E. coli Lambda exonuclease.Enzyme activity: Lambda exonuclease is a highly processive 5'→3' exodeoxyribonuclease, selectively digests the 5'-phosphorylated strand of dsDNA, exhibits low activity on ssDNA and non-phosphorylated DNA, and has limited activity at gaps in DNA [1, 2].Definition of enzyme activity unit: One unit is defined as the amount of enzyme required to produce 10nmol of acid-soluble deoxyribonucleotide from double-stranded substrate in a total reaction volume of 50μl in 30minutes at 37℃ in 1X Lambda Exonuclease Reaction Buffer with 1μg sonicated duplex [3H]-DNA.Purity: No exonuclease other than Lambda Exonuclease and free of endonuclease, RNAase, and phosphodiesterase.
Enzyme storage buffer： 25mM Tris-HCl, 50mM NaCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol, (pH8.0 at 25℃).
Inactivation or inhibition： Lambda Exonuclease can be inactivated by adding EDTA to a final concentration of at least 10 mM and incubating at 75℃ for 10 minutes.
Precautions： The Lambda Exonuclease has an optimal reaction temperature of 37℃ and can be completely inactivated by incubation at 75℃ for 10 minutes.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. Set up the following reaction on ice as follows:ComponentQuantity10X Reaction Buffer 5μl DNAXμl (up to 5μg) Lambda Exonuclease (5U/μl) 1μl (5U) Ultrapure Water Up to 50μl 2. Mix well by pipetting and centrifuge briefly to collect the liquid at the bottom of the tube. 3. Incubate at 37℃ for 30 minutes.4. Add EDTA to a final concentration of 10 mM to terminate the reaction. 5. Incubate at 75℃ for 10 minutes to inactivate Lambda Exonuclease. 6. Run the reaction products on an agarose gel or PAGE , and take photos to observe and analyze the digestion result.7. For further purification of the reaction products, it is recommended to use one of the following methods:a. Column purification. We recommend using 's PCR Clean Up Kit/DNA Purification Kit . b. Gel extraction. We recommend using 's DNA Gel Extraction Kit .c. Phenol-chloroform extraction followed by ethanol precipitation.FAQ:1. What is the difference between T5 Exonuclease and Lambda Exonuclease? Different from Lambda Exonuclease, T5 Exonuclease can degrade circular dsDNA with nicks as well as ssDNA much more efficiently. 2. What is the difference between T7 Exonuclease and Lambda Exonuclease?Different from Lambda Exonuclease, T7 Exonuclease acts at nicks in DNA and is less progressive. It has even lower degradation activity on ssDNA than Lambda Exonuclease. Because of its low progressivity, T7 Exonuclease may be more useful for unidirectional nested deletions. When using T7 Exonuclease, the reaction rate can be controlled by diluting T7 Exonuclease. In contrast, when using a highly progressive enzyme such as Lambda Exonuclease, dilution will produce a mixture of fully digested product and undigested substrate. 3. Can Exonuclease I be used in conjunction with a dsDNA nuclease to clean up plasmids?Exonuclease I can be used in conjunction with Lambda Exonuclease to clean up plasmids. Exonuclease III and T7 Exonuclease are also effective, but will damage plasmids with nicks. Although Exonuclease I can be used, we recommend using Nucleic Acid Exonuclease V (RecBCD) to remove chromosomal DNA from plasmids.
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