Labeling DNA Fragments
How are EMSA oligos labeled?
End-labeled oligos are used to generate IRDye® double-stranded DNA fragments.
- End-labeling is critical, because incorporation of dye into the DNA strand interferes with formation of DNA:protein complexes.
- Oligos are synthesized with the IRDye end label. Ready-to-use EMSA oligos are available from LI-COR, and custom oligos can also be generated.
- To generate a DNA duplex, forward and reverse oligos are required.
- BOTH oligos (forward and reverse) should be end-labeled with the same IRDye infrared dye. Signal intensity is greatly reduced (~70%) if only one oligo is labeled.
- Oligos must be annealed to form a double-stranded DNA fragment. To anneal, incubate the oligos in a 100 °C heal block for 5 minutes. Leaving the oligos in the heat block, turn it off and allow it to slowly cool to room temperature.
- IRDye EMSA oligos from LI-COR are pre-annealed.
What oligos are available?
Ready-to-use labeled oligos are available for a variety of common consensus sequences (see below). Custom oligos can also be used.
- Oligos MUST be end-labeled with IRDye infrared dye, and should be annealed to create a DNA duplex (see instructions above).
- IRDye oligonucleotides may be obtained from Integrated DNA Technologies (IDT)
Ready-to-use IRDye EMSA oligos:
EMSA Oligonucleotides Labeled with IRDye 700