| Description
Uracil DNA Glycosylase is recombinant from E. coli. Uracil DNA Glycosylase eliminates carryover contamination, which can result in false positives in PCR reactions. UDG catalyzes the cleavage of uracil base residues from both single- and double-stranded DNA, while leaving the DNA sugar-phosphodiester backbone intact. The resulting DNA is not suitable for use as a hybridization target or as a template for DNA polymerases. Uracil DNA Glycosylase will not remove uracil from RNA.
Quality Control
This product has passed the following quality control assays: functional absence of single- and double- stranded exonuclease as well as endonuclease and ribonuclease.
Control of PCR Carryover Contamination
Suggested changes to standard PCR protocol:
1. Substitute dUTP for TTP in PCR reactions.
Note: the potential contaminants must contain many uracil bases per molecule for the UDG protocol to be applicable (2).
2. Add 1 unit of UDG per 100 ¦Ìl of PCR reaction mix.
The UDG should be added as a normal part of the reaction mix prior to addition of mineral oil. One unit of UDG will eliminate up to 5 ng of contaminating uracil-containing DNA.
3. Incubate the PCR reaction at 37 ¡æ for 10 minutes prior to denaturation and amplification.
It is during this incubation that the uracil bases from carryover contamination are removed.
4. Increase the initial denaturation step of the first PCR cycle to 10 minutes at 94 ¡æ . This will
inactivate the UDG and break the contaminant DNA into small fragments.
5. Add two additional cycles to the amplification procedure. With some targets, it has been observed that incorporation of dUTP may be less efficient than TTP. The additional cycles will allow for normal yields of product to be obtained.
6. Maintain a final temperature of 72 ¡æ after amplification. Under special conditions, UDG has been observed to regain some catalytic activity after heat denaturation. By maintaining the PCR reactions at 72 ¡æ , UDG is completely inactive.
References
1. Lindahl, T., et al., (1977), J. Biol. Chem . 252 , 3286. 2. Longo, M., et al., (1990), GENE 93 , 125.
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