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I have desinged and tested primers for RT-PCR, then purified the PCR products from the gels and send them to sequence at GATC (SUPREMERUN) using the forward primers.

After blasting the reads to refseq, I see that they are ~30% shorter than the PCR product. Example:

PCR expected length = 182 bp Read length = 133 bp

I must mention that in the gels the expected PCR sizes were ok.

Does anyone have an idea what is the reason for this decrease in length?

Thanks

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Reliable sequencing reads start usually 50bp downstream from start of primers. Which means that 180bp-band (on the gel) sequenced with one primer will yield readable sequence in region 50-180bp (130bp length). Also I notice that sequences usually reported somewhat before reliable data, i.e. clean data starts after 52-55bp, but sequencing reports basepairs downstream from 48bp after primer start.

If you need whole-band sequence, you will have to sequence with set of two primers, each in different direction. Since you PCR this band, you already have your sequencing primers.

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