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Secondary structure has been a thorn in the side of many researchers since they discovered it existed. Sending in a sample only to see a truncated sequence at the end can be incredibly discouraging. Working with siRNA products can be even worse because getting through those samples is incredibly difficult.
No one likes getting their results back and seeing this:
Here are a few tips on how to improve the quality of this data:
1. Add DMSO. To improve the quality of secondary structure we usually add 1 microliter of DMSO. Best of all, DMSO does not degrade the quality of the reaction as a whole. If you are one of our customers, all you have to do is put add DMSO in the special instructions and we will do it to every sample on the order. This is also free of cost to you.
If this low-cost option does not work for you then I am afraid you might have to try something like:
2. If you are trying to PCR up a very G-C rich region, addition of betaine or DMSO to your PCR reaction can help, as can substitution of 7-deaza dGTP for 75% of the dGTP in your PCR reaction.
If this is still not working for you then:
3. Our special protocol (not disclosable for proprietary reasons): The second line of defense can be our special protocol. We have developed a protocol that can get through the most difficult secondary structures. It may be a slightly pricier option, but it certainly is worth it. We had tried many different reactions to get through the above example and by using this protocol we were able to achieve this:
Our turn around time remains the same for these samples.
If all else fails
4. Try manual radioactive sequencing as a last resort.
If you have any questions about this that need answering, feel free to contact support@etonbio.com. We work with Secondary Structure DNA Sequencing on a daily basis and we are more than happy to help out.
