Quality Controls for T4 DNA Ligase
Exonuclease contamination in a variety of T4 DNA Ligase samples as detected by capillary electrophoresis.
|Quality Controls for DNA Ligase
||Determined by SDS-PAGE analysis (or silver stained gel).
||Contaminating exonucleases are assayed by monitoring release of radioactivity labeled DNA substrate. Very low levels of specific types of exonuclease contamination are also assayed for using capillary electrophoresis with dye labeled substrate.
||Contaminating nucleases are assayed by incubation with HindIII fragments of lambda DNA.
||Incubation with supercoiled plasmid DNA for 4 hours in used to detect any nicking or non-specific nuclease degradation.
||Room temperature ligation is performed.
Capillary electrophoresis testing of T4 DNA Ligase and other commercially available ligases. Reactions contained 20 nM fluorescent labeled substrate, 1X T4 Ligase Buffer, and 5 µl enzyme in a total reaction volume of 50 µl. Reactions were incubated at 37°C for 16 hours followed by heat inactivation at 80°C for 20 minutes. 1 µl of each reaction was combined with 10 µl of Hi Di™ Formamide containing GeneScan™ 120 LIZ® Size Standards and injected on the 3130 x 1 Genetic Analyzer. Three double-stranded substrates were tested and the data indicates whether the degradation occurs on the top or bottom strand.
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