I am attempting to ligate an oligonucleotide with a gene that has a double-stranded DNA sequence. Approximately 10-20% of the DNA forms gene dimers instead of ligating with the oligonucleotide. This dimer formation is nonspecific, occurring between identical gene ends, resulting in ligation between non-complementary cohesive ends. The ligation is performed at room temperature (RT), 37°C, for 1 hour. How would you recommend improving the ligation fidelity?
Thank you.
If you increase the oligo to fragment ratio you should drive more of the reaction towards the product you want.
Hello,
can you be more specific on your setup ?
- insert length, insert to gene ratio used for ligation, oligonucleotide length ?
- what are the ends sequence you try to join with the T4 ligase ?
- the ligation is done for 1 hour at RT or 37°C ? or both stepwise ?
Regardless, something that always increase my ligation success was to do the ligation with increased temperature, start at 16°C then 20 then 24 then ... until you reach 37°C. Over the course of two hours.
You can also try 16°C overnight .