T4 DNA Ligase (D2886) catalyzes the formation of a phosphodiester bond between the terminal 5′ phosphate and a 3′ hydroxyl groups of duplex DNA or RNA. The enzyme efficiently joins blunt and cohesive ends and repairs single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.1 The protein source is a recombinant E. coli strain carrying the cloned T4 DNA Ligase gene.
Common applications for T4 DNA include the following:
One T4 DNA Ligase cohesive end unit is equivalent to approximately three cohesive end units as measured with a Lambda-Hind III DNA fragment substrate in 1X T4 DNA Ligase reaction buffer.
One Weiss Unit is approximately equivalent to 22 units.
T4 DNA Ligase is ATP dependent. For best results, store the reaction buffer at -20 °C and discard after one year of storage.
Single-insert ligations are optimal when targeting an insert:vector ratio from 2 to 6. A ratio above 6:1 will promote the insertion of multiple fragments, and ratios below 2:1 will reduce ligation efficiency.
One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of P32 from pyrophosphate into ATP as Norit-absorbable material in 20 minutes at 37 °C.
1. Add all of above components to a clean reaction vessel, mix well by pipetting.
2. Incubate at 25 °C for 30 minutes.
3. Immediately purify DNA using PCR clean-up column and elute in ~50 µL.
4. OR - Immediately dilute (at least 1:10, but enough such that 0.1-10 ng of ligation product will be transformed) in TE or water
5. Transform 0.1-10 ng of ligation product into chemically or electrocompetent cell line that is compatible with vector
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