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Uracil-DNA Glycosylase

for prevention of carry-over contamination in PCR Cat. No. 1 444 646 100 units
Cat. No. 1 269 062 25 units Description Uracil-DNA Glycosylase (UNG) (from E. coli) catalyzes the removal of uracil from single- and double-stranded DNA that has been synthesized in the presence of dUTP. The apyriminic sites formed by UNG are susceptible to cleavage by heat under alkaline conditions (carry-over prevention). UNG will not digest 3'-terminal dU, dUTP, primers labeled with biotin-dUTP, or digoxigenin-dUTP. Ribouracil residues in RNA are also unaffected by UNG. Application Prevention of carry-over contamination in:
  • PCR with Taq DNA Polymerase.
  • RT-PCR with C. therm. Polymerase One-step RT-PCR System.
Operating parameters
  • Substrate: Single- or double-stranded DNA containing deoxyuracil (DNA synthesized in the presence of dUTP).
  • Incubation conditions for cleavage of uracil-glycosidic bonds: 20C, 110 min.
  • Thermal stability (pH 8.3): half-life at 40C, 2 min; half-life at 45C, 0.5 min.
  • MgCl2 concentration: 2.5 mM.
  • dNTP concentration: 200 M dNTPs; 600 M dUTP.
  • Conditions for inactivation of enzyme: 95C, 2 min.

Notes:

UNG (2 units) will degrade up to 106 uracil-containing DNA molecules in 10 min. In contr ast to the enzyme from E. coli, heat-labile UNG will not modify dU-containing PCR products for at least several hours at 4C. Therefore, it is not necessary to freeze dU-containing PCR products immediately after amplification, or to hold the reaction mixture at 72C until it is used. However, note that after prolonged storage (>8 h) residual enzyme activity may lead to degradation of PCR product. For long term storage, PCR product should therefore be frozen at 20C.

Key advantages
  • Prevents carry-over contamination in PCR, because the enzyme modifies all uracil-containing products from previous PCRs. These products are then hydrolyzed by heat under alkaline conditions.
  • Does not degrade native (thymidine-containing) templates, because the enzyme is free of contaminating exo- and endonucleases.
  • Does not interfere with PCR amplification, because the enzyme is not active at temperatures typically used for PCR applications.

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Related biology technology :

1. Preventing Carry-over Contamination with Uracil-DNA Glycosylase
2. Uracil-DNA Glycosylase, heat-labile
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