Hi! i am doing IEF using Bio-Rad IPG strip 17cm and IEF system, One thing which always bothers me is voltage ramping, once the rehydration is over after placing wicks IEF is started but somehow voltage ramping does not match with the time say from 500Volts to 10000 volts in 6 hours but voltage never reached 10000 even after 6 hours. What could be problems besides high salts which is not present and rehydration is in standard active rehydration buffer.How do we know time requried for focusing how do we know that this is overfocusing, i can't find anywhere.Eagerly waiting for suggestion and advice.
thanks.
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