This is my first genomics project and I have been collaborating with several other scientists. I have amplified 720 PCR products, and I am preparing to purify them and send them in for sequencing, as the sequencing core I am sending them to does not offer clean up services. My plan was to use Exonuclease I and Shrimp Alkaline Phosphatase enzymatic cleanup and I have already purchased these from New England Biolab (M0293L and M0371L). I have used all my budget money and cannot afford to buy a column purification kit. Will the samples still be alright for Sanger sequencing with the blue and yellow dye? This didn't occur to me as a problem before because the documentation says "The dyes in the master mix do not interfere with PCR performance and are compatible with downstream applications such as DNA sequencing, ligation and restriction digestion," but later it recommends colorless mix for analysis by fluorescence excitation and absorbance, as Sanger capillary would be. Does anyone have experience with this? I would rather delay my project than send in all 720 samples only to discover the sequencing repeatedly fails.
As the manufacturer states that it is compatible with downstream sequencing I would believe them. Even if the dye runs within the sized sequence it is usual that the bases are readable under the dye peak except for the one colour that is nearly the same colour as the dye and that shows as a missing base. Also the pcr product is only a fraction of the whole pcr mix and in sequencing many cycles are run increasing the peak intensities with each cycle.
I think that it will work but if you are unsure then just send them a couple of samples to sequence. Most companies in this situation would sequence a couple of samples free if an order for 700+ more samples was likely to result.Speak to your company representative about running some free samples and a discounted price for the upcoming large sequencing order.
I think that using exonuclease and Shrimp alkaline phospharase (as a combination or separately) will be effective in cleaning up the PCR products through removing the extra dNTPs and primers in the mixture. I think it will work. I myself used the ExoSap-it in minisequencing (primer extension) experiments previously... It was effective
Good luck
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