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unpredictable plasmid - can digest but not sequence. . . (Oct/17/2005 )

We are having a strange problem in our lab. We have three constructs in the pUHD10-3 hygro backbone that we can digest to see the inserts but every time we send them out for sequencing we get errors saying that they were unable to prime off of the DNA or that there wasn't any DNA in the tube. blink.gif We have been able to use our sequencing primers successfully for PCR and we have sent out other constructs in this backbone for sequencing before with no problems. We're kind of baffled. Has anyone else run into this before?

The plasmids have been prepped using both the Qiagen and Promega Wizard kits, all with the same kooky results.

Thanks for any ideas!! biggrin.gif


Some regions can be difficult to sequence, partically regions with high G+C content. Perhaps try sequencing on the other strand or contact your sequencing lab for tips on sequencing through difficult regions.


Another possibility is that you have carry over of ethanol from the miniprep....i had a problem like that once and the sequence lab told us that minimum quantity of EtOH will prevent sequencing


using promega columns, i get clearer results by let the column dry after ethanol wash on the bench (no heating) and for at least 30'...
check also the concentration of your prep. I noticed that less than 400ng/┬Ál is reducing chances of sequencing.


thanks for the ideas! biggrin.gif