sequencing - high peaks, then low peaks (Jun/21/2005 )

Hi,

I have recently sequenced several clones using an automated
sequencer.
It was OK when I used the same machine one month ago, I could read about 400 bp.

However, this time the peaks were very high for the first 100 bp,
then they became very low and difficult to read.

Could anyone suggest where the problem lies?

Have you tried adjusting the vertical scale? Most program allow you to do that such as this one http://www.protocol-online.org/forums/inde...?showtopic=7208

Low peaks do not necessarily mean bad base call.

Thank you, pcrman.
I'll try it.

This is often a symptom of too high a concentration of template DNA. With large amounts of template, the dNTPs and ddNTPs in the sequencing mix are used up rapidly in early stages of the reaction. Try reducing your template concentration by a factor of 10.