-dilution of "Big Dyes"
Woodland, M P
m.p.woodland at ic.ac.uk
Wed Dec 20 04:35:22 EST 2000
Hi - I use a 377 in a small core facility and routinely dilute
the BIGDYE v2.0 by 2x and use a 10ul
reaction volume and purify the sequencing reactions with
Amersham/Pharmacia spin columns.
To gain maximum reads I use the ABI Long Read protocol. - I
could'nt afford to use this set of
protocols unless I diluted the BIGDYE ! For the m13 control just
this Friday I achieved just under IKbp
with 4Ns - this is not unusual - not always the case foe samples.
If a sample sequences poorly I use
the std protocol- although this rarely appears to make a great
difference. One aspect of the v2.0 is that the front end is much
cleaner. I only use DMSO
with GC rich as I have always understood that TAQ pol does'nt
like it. When the CORE kits were available
I always use to add more TAQ halfway thru the GC rich cycles to
extend the read length.
What is different about capillary based systems that dillution and
v2.0 apparently gives
such poor results?
Uni. of London
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