Hi everyone!
I would like to know if an exonuclease I treatment after isolating DNA samples from an agarose gel is enough for sequencing. I know that it is recommended to use ExoSap which includes also alkaline phosphatase, but i wonder if exonuclease is capable of removing all primers and dNTPs by its own.
Thanks!
exo will not remove the ntps from the pcr mix where they are in huge excess. The ratio of ddNTP and dNTP in sequencing reaction mixes is tightly controlled to allow sequencing of the correct length so leaving more ntps in the template sample will cause the sequencing to start later so for example if your sequence usually starts at about 40 bases from the sequencing primer then without the SAP stage to remove the ntps I would expect your sequence to start at 100 bases from the sequencing primer so I would always do the SAP part of the exo/sap procedure. As for the other part of your question re primer removal exo sap is very good at cleaning up primers but occasionally there is a group of primers that the exo cannot degrade and sometimes this gives you a poor sequence from one end of the template but this is quite uncommon
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