Sample stability on 3700 deck.

Paul Shinn pshinn at mail2.sas.upenn.edu
Tue Oct 26 07:28:14 EST 1999

Phillip San Miguel (pmiguel at purdue.edu) wrote:
: Is there any consensus on how long samples can be left on
: the 3700 (without covers--we haven't got the fang piercers
: yet.) Could light be causing the sample degradation? Do
: different sequence reaction chemistries cause different
: effects here?

    Currently, I use only 96 well plates so 4 plates is the most I can run
at once.  I always run the big dye primers before the big dye terminators
and I never run more than 2 BDPs in one sitting.  I have never been able
to get good sequence from the 3rd plate of BDPs so I'm going to say from
my experience that BDPs resuspended in 10ul 50:50 water:formamide will
last 6 hours on the deck (time from hitting Run button to time of load of
2nd plate).  The last plate of BDTs is never as good as the first or
second as evidenced by G dropouts.
    I have been able to rerun a plate of terminators after it's sat on the
deck over night but usually this only works if the reactions were strong
to begin with.  Our normal shotgun chemistries are 1/8 BDTs and 1/4 BDPs
off M13.  Both sets are resuspended in 10ul 50:50 water/formamide (EDTA). 
I'm using the modified Low Temp run module for terminators.  When we have 
good M13 templates, our read lengths are consistently well above spec--by 
eye at least.  I can't be 100% sure yet because phred isn't trained for 
3700 data yet.
    I'm excited about the fang release, too.  I'm even more excited about 
POP5, though!


Paul Shinn    
Sequencing Coordinator                                    ,___o
pshinn at neomorph.bio.upenn.edu                            _-\_<,
Arabidopsis thaliana Genome Center                      (*)/'(*)
(215) 573-7256

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