Hi everyone,
I am working on a hybridization chain reaction (HCR) and trying to visualize it with gel electrophoresis. The visualization of the HCR works but for the single components of this amplificaion strategy I get something, I wouldn't expect. Does anyone know why I have two bands when only adding a hairpin sequence of 48 bp (H1 and H2)? I would assume it results in only 1 band as I have with my initiator, DP. Can this difference occur purely based on the folding-status of the DNA sequence?
i'm not very familiar with this technique but I think that the larger bands (there are three and some smearing) would be due to the hairpins concatamerizing. if they form a hairpin to themselves then they could dimerize or more. do you heat/cool the hairpins before running the gel? perhaps try running the gel at a lower or higher voltage (use sodium borate buffer for higher voltage). this might not be an issue if the HCR is working just some of your hairpins will bind each other rather than what they are supposed to.
Ah yes, of course. I didn't think of the self-hybridization. For now, I work at room temperature so next I'll try to heat up the sequences and run it at different voltages to see if it makes a difference. This is indeed important for the HCR as well. Thank you!
The presence of two bands for the single hairpin sequences (H1 and H2) on your gel electrophoresis can be attributed to different conformational states of the hairpins. Here are some potential reasons for this observation:
In your gel image:
- Lane 1 (5 µM H1) and Lane 2 (5 µM H2) both show two bands, likely indicating the presence of different conformational states or potential dimerization.
- Lane 3 (2.5 µM H1 + 2.5 µM H2) shows a smear, suggesting hybridization and formation of HCR products.
- Lane 4 (5 µM DP 25) shows a single band, indicating a uniform conformation without significant secondary structure or dimerization.
To further investigate the reason behind the two bands, you could consider:
- Running the gel under different conditions (e.g., temperature, buffer) to see if the band pattern changes.
- Treating the samples with denaturing agents (e.g., urea) to disrupt secondary structures and running them on a denaturing gel.
- Performing a control experiment with known hairpin sequences to compare the migration pattern.
This should help you determine whether the observed bands are due to secondary structure, partial folding, or other factors.
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