Can complementary RNA and DNA anneal or hybridize naturally at room temperature or should I denature and cool them as we do for annealing DNA oligos?
Should I add reagents like DMSO or MgCl2 etc to aid in annealing process.
Hi.
I will try a protocol using an annealing buffer. You can use a similar protocol for DNA and RNA keeping in mind that the idea is to disrupt any secondary structure within each oligonucleotide.
Here the annealing buffer composition:
Annealing Buffer Composition (1X)
10 mM Tris, pH 7.5 - 8.0
50 mM NaCl
1 mM EDTA
Thank you, have you tried annealing RNA-DNA oligo? If so, how to confirm that they have annealed? Is it by gel electrophoresis?.
Hi Shalini,
I see two options:
1. nucleic acids display hyperchromic effect (the increase of absorbance (optical density) when they are single as compared to doubale stranded)
so, use a nanodrop to compare the absorbance of both annealed and single stranded (simple mixture of both oligos).
2. Run a bit of each oligo in isolation and expected annealed oligo on 2% agarose gel with Ethidium bromide. EtBr bind better to dsDNA than ssDNA, so you should see higher intensity if it works...
Can I add 2' O Methyl modification to one of the duplex to increase hybridization?
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