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CleanSEQ on the ABI 3100

ubjecting samples eluted in water to evaporation,

degradation and the appearance of dye blobs in sequence traces. We tested a number of CleanSEQ elution buffers and procedures in an effort to inhibit sample evaporation and prevent sample degradation, thereby improving sequence quality on the 3100 (data not shown). As result of these studies, we recommend the elution of samples from CleanSEQ microparticles using water followed by application of mineral oil.

We collectively ran over 1000 samples using our recommended oil overlay protocol and found significantly increased signal strengths and extended protection of samples from degradation compared with formamide elution. Importantly, no decrease in capillary performance was observed due to mineral oil usage. This simple procedure results in high quality sequence reads and eliminates the appearance of dye blobs (Figure 2).


Experimental Methods
pGEM was sequenced using v3.1 BigDye Terminator chemistry at 1/4x dilution. Sequencing reactions were purified using the standard CleanSEQ 96 protocol. Samples were eluted in either 50l of 100% formamide, 50l of water, or 50l of water followed by application of 20l of mineral oil. No sample transfers were required* from the time of sequencing set up through loading onto the 3100 with POP6 polymer. *Plate transfer required when using POP4 polymer.


Higher Signal Strengths using Water Elution
We compared signal strengths and signal to noise ratios for samples eluted in water, water with mineral oil application and formamide. Signal strengths are significantly higher using water elution com
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