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Lambda exonuclease

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Lambda exonuclease is a DNA-specific exonuclease enzyme that progressively digests DNA from the 5' end in the 3' to 5' direction. It catalyzes the removal of mononucleotides from the 5' end of double-stranded or single-stranded DNA.

Automatically generated - may contain errors

Lab products found in correlation

Phi29 polymerase, by New England Biolabs (2 mentions) Ampligase, by Illumina (2 mentions) Taq polymerase, by Roche (1 mentions) Sodium chloride (nacl), by Avantor (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Kh2po4, by Merck Group (1 mentions) Na2hpo4, by Avantor (1 mentions)

3 protocols using lambda exonuclease

1

DNA Fragment Circularization and Amplification

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Example 1

A protocol suitable for performing the method illustrated in FIG. 1 is as follows: 1) 10 ng of DNA is digested with 1 unit of restriction enzyme in corresponding compatible restriction enzyme buffer. The reaction is incubated in 37 C for 1 h, followed by enzymatic deactivation at 80 C for 20 min. 2) The DNA fragments are denatured to single stranded fragments at 95 C for 10 min and mixed with probes and ligase to form circles. The probe pool are added in 10 pM individual concentration along with 1 U of Ampligase (Epicentre) and incubated at 55 C for 1 h in ligase buffer. 3) 1 U Exonuclease is added to remove non-reacted probes and fragments. I U of Lambda exonuclease (Epicentre) is added at 37 C for 1 h in corresponding exonuclease buffer followed by enzyme inactivation at 80 C for 20 min. 4) The remaining circles are amplified by RCA. 1 U of phi29 polymerase (New England Biolabs) is added in corresponding phi29 buffer and nucleotides (dNTPs) at 37 C for 1 h.

US10731214B2. Nucleic acid probe and method of detecting genomic fragments (2020-08-04). Vanadis Diagnostics [SE]. Inventors: Carl Oscar Fredrik Dahl [SE], Olof John Ericsson [SE].
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2

DNA Extraction and Amplification Protocol

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Na2HPO4, NaCl, and ethylenediaminetetraacetic acid (EDTA) were obtained from VWR International (Vilvoorde, Belgium). KH2PO4 was bought from Sigma-Aldrich (St Louis, MO, USA). Proteinase K from Invitrogen was purchased from Life Technologies (Carlsbad, CA, USA). TRIS (tris(hydroxymethyl)aminomethane), sodium dodecyl sulfate (SDS), and Sephadex G-50 medium were bought from GE Healthcare Europe GmbH (Freiburg, Germany). Lambda exonuclease from Epicentre was acquired from Westburg (Leusden, the Netherlands). Deoxyribonucleotide triphosphate (dNTP), polymerase chain reaction (PCR) buffer, and Taq polymerase were obtained from Roche Diagnostics (Basel, Switzerland). All primers and synthetic DNA sequences were purchased from Integrated DNA Technology (Leuven, Belgium). Lysis buffer (1.2 g/L TRIS, 23.3 g/L NaCl, 0.7 g/L EDTA) and phosphate-buffered saline, 10 times concentrated (10× PBS) (1.29 M NaCl, 0.05 M Na2HPO4 • 2H2O, 0.015 M KH2PO4, pH 7.2) were made in our laboratory.
Vanden Bon N., van Grinsven B., Murib M.S., Yeap W.S., Haenen K., De Ceuninck W., Wagner P., Ameloot M., Vermeeren V, & Michiels L. (2014). Heat-transfer-based detection of SNPs in the PAH gene of PKU patients. International Journal of Nanomedicine, 9, 1629-1640.
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3

DNA Circularization and Rolling Circle Amplification

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Example 1

A protocol suitable for performing the method illustrated in FIG. 1 is as follows: 1) 10 ng of DNA is digested with 1 unit of restriction enzyme in corresponding compatible restriction enzyme buffer. The reaction is incubated in 37 C for 1 h, followed by enzymatic deactivation at 80 C for 20 min. 2) The DNA fragments are denatured to single stranded fragments at 95 C for 10 min and mixed with probes and ligase to form circles. The probe pool are added in 10 pM individual concentration along with 1 U of Ampligase (Epicentre) and incubated at 55 C for 1 h in ligase buffer. 3) 1 U Exonuclease is added to remove non-reacted probes and fragments. I U of Lambda exonuclease (Epicentre) is added at 37 C for 1 h in corresponding exonuclease buffer followed by enzyme inactivation at 80 C for 20 min. 4) The remaining circles are amplified by RCA. 1 U of phi29 polymerase (New England Biolabs) is added in corresponding phi29 buffer and nucleotides (dNTPs) at 37 C for 1 h.

US10240198B2. Nucleic acid probe and method of detecting genomic fragments (2019-03-26). Vanadis Diagnostics [SE]. Inventors: Carl Oscar Fredrik Dahl [SE], Olof John Ericsson [SE].
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