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Now,I meet a commom DNA sequencing problem.The trace has In my test,I used Millipore filter plate ,Pall filter plate and EXOSAP to clean up PCR products. The sequecing results which purified by Millipore and Pall filter plate were good. But the trace of those which purified by EXOSAP had one or more large, broad peaks between bases 50 and 140 as you have mentionded in your website.I dont’t think it was caused by Poor post-sequencing clean up or Lost pellet because I used same method to clean up sequencing products.I will be appreciated of your help!
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This problem (ie dye peaks) may still be caused by a clean up problem even thought it doesn’t seem possible at first. The reason for this is the exosap method does not remove salt from the reaction (in particular MgCl2). If you clean up the reaction using ethanol precipitation then the extra salt present from the exosap will cause the leftover dye to precipitate.
The solution to this problem is to reduce the amount (or concentration) of ethanol used in the clean-up. Try adjusting by a few percent at first because it is easy to go too far and lose the pellet.
Daniel
Daniel Tillett
Nucleics Support
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