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I’m new to sequencing, and I am having major issues with my sequences. I have purified my PCR product using a standard PCR purification kit, and had it sequenced. The first part of the sequence was what I expected and where it should end, it doesn’t. Instead it carries on and I have no idea why it carries on. I had run the PCR product on an agarose gel and there was only one amplicon. The PCR product size is pretty small (~90 base pairs) and I don’t know if it’s because of the product size or something wrong with my primers. Any help and advice would be greatly appreciated.
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You can have low level PCR product contamination in what looks like a single product. What happens is the primer gets to the end of your 90bp product and then the sequencing product for it ends. The signal will drop right down to a very low level and continue off the contaminating product. Even though this might only be 1/1000th the level of the main product there is enough of it there to give a sequencing signal once the main product ends. You don’t see this in the first 90bp because the signal level is too low, it is only when teh stronger signal ends that this background signal is observed.
Daniel Tillett
Nucleics Support
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