New England Biolabs, M0701S, Thermostable Endonuclease Q

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to cleave 1 pmol of a 60-mer oligonucleotide ssDNA containing a single deoxyxanthosine site* in a total reaction volume of 20 μl after 1 hour at 65°C. *A deoxyxanthosine site is synthetically prepared with dX at the 24th position of a 5´ FAM-labelled 60-mer ssDNA oligo. Reaction Conditions 1X NEBuffer™ r2.1 Incubate at 65°C 1X NEBuffer™ r2.1 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 500 mM NaCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No FAQ Q: What is the difference between the suite of USER® enzymes (#M5505, #M5508, #M5509) and Thermostable Endonuclease Q (#M0701)? A: The USER products (USER Enzyme (#M5505), Thermolabile USER II Enzyme (#M5508) and Thermostable USER III Enzyme (#M5509) are a mixture of two enzymes: UDG and an endonuclease. In contrast, Thermostable Endonuclease Q (#M0701) is a single enzyme. Although the USER enzymes and Thermostable Endonuclease Q both recognize and cleave at deoxyuracil in DNA substrates, the resulting products are different. The end products of the USER enzymes are one-nucleotide gaps with varying functional ends based on the type of USER implemented; the deoxyuracil is removed. The products of Thermostable Endonuclease Q are nicks with a 3′-OH and a 5′-phosphate; the deoxyuracil is still present on the 3′ segment of DNA. Q: What is the difference between Endonuclease V (#M0305) and Thermostable Endonuclease Q (#M0701)? A: Although both Endonuclease V and Thermostable Endonuclease Q recognize deoxyinosine in DNA substrates that result in the formation of a nick with a 3′-OH and a 5′-phosphate, the location of the nick is different. Endonuclease V cleaves the second phosphodiester bond on the 3′ side of the deoxyinosine; Thermostable Endonuclease Q, cleaves on the phosphodiester bond immediately 5′ to the deoxyinosine. In addition, Endonuclease V activity is optimal at 37°C and can be heat-inactivated; Thermostable Endonuclease Q activity is optimal between 65°C and 85°C and cannot be heat-inactivated. Q: What is the activity of Thermostable Endonuclease Q (#M0701) in different NEBuffers? A: table, th, td { border: 1px solid black; border-collapse: collapse; } table, td{ border: 1px solid black; border-collapse: collapse; } Thermostable Endonuclease Q has activity in the following buffers (relative to activity in NEBuffer r2.1): NEBuffer r1.1 50% NEBuffer r2.1 100% NEBuffer r3.1 100% rCutSmart Buffer 75% ThermoPol® Reaction Buffer 100% ThermoPol II (Mg-free) Reaction Buffer 25% LongAmp® Taq Reaction Buffer Pack 75% Isothermal Amplification Buffer 50% OneTaq Standard Reaction Buffer 75% Standard Taq Reaction Buffer 50% Q5® Reaction Buffer 50% Q5U® Reaction Buffer 75% Q: What is the molecular weight of Thermostable Endonuclease Q (#M0701)? A: The molecular weight of Thermostable EndoQ is 48,515 Daltons. Q: Is Thermostable Endonuclease Q (#M0701) a tagged protein? A: Thermostable Endonuclease Q has a C-terminal His tag. Q: Can the product of the Thermostable Endonuclease Q (#M0701) reaction be ligated? A: Yes, the product of a Thermostable Endonuclease Q reaction can be ligated. The enzyme creates a nick, resulting in a 3′-OH and a 5′-phosphate. Q: How close to the 5′ DNA end will Thermostable Endonuclease Q (#M0701) cleave? A: Thermostable Endonuclease Q can cleave at deoxyuracil, deoxyinosine, and deoxyxanthosine as close as from the third position of the 5′-end in both ssDNA and dsDNA. Q: How close to the 3’ DNA end will Thermostable Endonuclease Q (#M0701) cleave? A: At a minimum, Thermostable Endonuclease Q can cleave at deoxyuracil, deoxyinosine, and deoxyxanthosine starting at the sixth position from the 3′-end of either ssDNA or dsDNA. Q: What temperatures is Thermostable Endonuclease Q (#M0701) active? A: Thermostable Endonuclease Q is active between 37 – 90°C, but has optimal activity between 65°C and 85°C. Temperature profile Thermostable Endonuclease Q is active over a wide range of temperatures. Thermostable Endonuclease Q (#M0701, 0.2 U) activity was tested on 1 pmol of a fluorescently-labeled single-stranded oligonucleotide containing a single uracil for 1 hour in 1x NEBuffer r2.1 at various temperatures. Cleaved products were detected by capillary electrophoresis. Thermostable Endonuclease Q is active over a wide range of temperatures (37°C – 90°C), but displays optimal activity between 65°C and 85°C. Q: What modified bases will Thermostable Endonuclease Q (#M0701) recognize and cleave? A: Thermostable Endonuclease Q specifically recognizes and cuts at deoxyuracil, deoxyinosine, deoxyxanthosine, and abasic sites in both ssDNA and dsDNA. Additionally, Thermostable Endonuclease Q will recognize and cut at ribouracil within a DNA strand. Q: Does Thermostable Endonuclease Q (#M0701) have activity in DNA-RNA hybrid duplexes? A: Thermostable Endonuclease Q can cleave deoxyuracil within DNA strands when the complementary strand is either DNA or RNA. Additionally, Thermostable Endonuclease Q will recognize and cut at ribouracil within a DNA strand when the complementary strand is either DNA or RNA. Thermostable Endonuclease Q cannot cleave when either a deoxyuracil or ribouracil is within an RNA strand. Q: What is the relative activity of Thermostable Endonuclease Q (#M0701) on different modified nucleobase-containing substrates? A: Relative Activity of Thermostable Endonuclease Q on Various DNA Substrates: Q: Do any of the USER enzymes leave ligatable ends when cleaving at a Uracil? A: No, neither USER Enzyme (NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) nor Thermostable USER III Enzyme (NEB #M5509) will leave ligatable ends when cleaving at a uracil. However, Thermostable Endonuclease Q (NEB #M0701) will nick 5´ to a uracil (leaving the nucleobase intact), but leave ligatable 3´ OH and 5´ P ends after cleavage. Q: How close to the ends of DNA can Thermostable Endonuclease Q (NEB #M0701) cleave? A: Thermostable Endonuclease Q will cleave a deoxyuracil 6 base pairs or nucleotides from the 5’ end of either double-stranded or single-stranded DNA. Thermostable Endonuclease Q will cleave a deoxyuracil 3 base pairs or nucleotides from the 3’ end of either double-stranded or single-stranded DNA. Please refer to Cleavage of deoxyuracil close to the ends of dsDNA and ssDNA for more information.