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sequencing question help! - urgent! (May/13/2010 )


So I made an error in the lab. Normally, when my lab sequences plasmids for other people (by sanger sequencing), we use 10ng/ul of plasmid with 5uM of their plasmid sequencing primer in the sequencing reaction. However, today I made the mistake of using 20uM of their plasmid sequencing primer. I realized after the sequencing reaction went to completion on the thermocycler.

My question is do you think, generally, I added in too much plasmid primer such that the data will look bad after sequencing reaction cleanup? What is the common experience for this kind of situation? Is there anyway I can fix this before I do the cleanup step?


I think it still might be OK. Four-fould increase in primers should not be a problem.
Increasing primer concentration effectively decreases melting temperature but as long as this primer is unique or specific enought it will work correctly.


If you use excess of primers, it's probable, that you will get stronger signal on the beginning of the sequence and low on the end. That could mean two possible problems, first the signal in the begining could be overloaded and thus impossible to read (that depends on the sensitivity and dynamic range of the sequencer you use) and second the read itself would be short (which doesn't matter you your sequencing product is short, like 300 bp or so).
Anyway, there is nothing much you can do about it now (only maybe dilute the cleand-up product if you get too high signal on the beginning).