rM. Thermoautotrophicus TDG with Buffer

4070-500-EB Datasheet / COA / SDS

Discontinued Product

4070-500-EB has been discontinued.
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rM. Thermoautotrophicus TDG with Buffer Summary

Kit containing rM. Thermoautotrophicus TDG and a buffer.

Product Description

TDG is a thermostable thymine DNA glycosylase from Methanobacterium thermoautotrophicum. The optimal temperature for the enzyme is 65°C. The enzyme lacks significant AP lyase or endonuclease activity. TDG works effectively in heteroduplex analysis to detect C to T transitions.

TDG enzyme recognizes T/G mismatches in duplex DNA and cleaves the strand with the T. The opposite strand is not cleaved. The enzyme also recognizes G/G mismatches if at least one nearest neighbor is an A or T and nicks one strand or the other. The enzyme exhibits poor AP lyase activity. One Unit is the amount of enzyme required to cleave 1 pmole of an oligonucleotide duplex containing a T/G mismatch in 1 hour at 65 °C. Only the strand containing the T is cleaved.

Assay Conditions and Analysis

4 pmoles of T/G mismatch oligonucleotide set with the T oligo end-labeled, 1X REC Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 10 mM EDTA), and serial dilutions of enzyme in a 20 μL reaction volume are incubated for 1 hour at 65 °C. To complete cleavage of a basic site, fresh 1N NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95 °C. For analysis, 24 μL of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added, and the samples heated at 95 °C for 10 min then fast cooled to 2-8 °C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

Kit Contents

• 10X REC™ Reaction Buffer 4
• Thermostable TDG Enzyme

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended on the product label.
Storage
Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
N/A

Limitations

For research use only. Not for diagnositic use.

Product Datasheets

Reagents Provided

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