I have been using BigDye V3.1 to do my sequencing. There is always a region at abt 190bp that i cant call out my base reading. I'm wondering if it is caused by ethanol contamination. what is the best way to preciptate my product after the BigDye Terminator Reaction?
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Leyre
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Posted 02 May 2004 - 08:14 AM
Hi! this is the protocol I use to precipitate DNA with etanol and EDTA (it works for the BigDye 3.1)
20 microl. of your sequencing reaction plus 5 microl. of 125 mM EDTA
Add 60 microl. of etanol 100%
Vortex
RT 15 minutes
centrifugue 20 minutes at 14.000 rpm 4ºC
Remove the etanol
Add 200 microl. of etanol 70%
centrifugue 2 minutes at 14.000 rpm 4ºC ( make this 2 times more)
And then remove all the etanol, make sure that there's no etanol.
I'll hope it works for you!!
Bye!
20 microl. of your sequencing reaction plus 5 microl. of 125 mM EDTA
Add 60 microl. of etanol 100%
Vortex
RT 15 minutes
centrifugue 20 minutes at 14.000 rpm 4ºC
Remove the etanol
Add 200 microl. of etanol 70%
centrifugue 2 minutes at 14.000 rpm 4ºC ( make this 2 times more)
And then remove all the etanol, make sure that there's no etanol.
I'll hope it works for you!!
Bye!
