|
Update 6/4/96 We have been running Long Ranger gels for the past four months. The reliablity of the gels has increased and I would suspect htat we never see the fast-fade problem again. We were at first hesitant to change to Long Ranger because everything was working but had seen evidence that read lengths would improve. We got that and a more reliable gel. It's easy: go with the identical conditions you currently use but use the Long Ranger at 1% higher concentration. That's it.
Old post follows:
__________________________
I joined this newsgroup just after I had posted a problem to the ABRF and bionet bulletin boards. Since the problem has just occurred again I am posting the original post and the summary of responses to this board for suggestions.
To attempt to make it as coherent as possible I will rearrange it in chronological order with new comments placed in [brackets].
Thanks, Paul
Paul Morrison Dana1030
Molecular Biology Core Facilities
Dana Farber Cancer Institute
1 Jimmy Fund Way
Boston, MA 02115
paul_morrison@dfci.harvard.edu
_________ORIGINAL POST __________________ORIGINAL POST________________
Subject: ABI 373A DNA Sequencer data just fades away.
Here is a problem that is starting to really bug me. It has been
occurring intermittently since Oct94 at an increasing rate. Just as
soon as we think we have figured out the problem. Whap, it's back. So a
brief description of the problem.
"Core Facility" (different sample preps) templates sequenced double
stranded, Taq polymerase, cycle sequencing, DYE labeled terminators.
On occasion we will get a gel that has _something_ wrong with it. We
now call them "fast-fade" gels. On these fast-fade gels many lanes of
data will look good but then abruptly (within 20 nucs) fade to
practically nothing. These problem lanes will usually (but not all)
come in groups on the left or right hand side of the gel. This problem
has occurred in various places. After 50 to after 400 nucs of good
data. We have contemplated just about every reason and not one of them
holds up. Because it happens infrequently and we are sequencing Core
Facility DNA from various sources this has been very hard to track
down.
[Erratic occurence, 1 every ten days or so.]
What the problem looks like on the:
Analysis file. Peaks are great and then abruptly drop and get the
"shakes". There is usually trailing _in front_ of the peak that happens
as the peaks spread out and quickly change to shaky waves of color.
Raw Data file. Peaks are great and then fade to nothing.
Gel file. Peaks are great and then get blurry. It looks as if the same
amount of color is there but the resolution has gone to pot.
[Very difficult to diagnose whether the signal strength or the resolution takes the hit]
Reasons why we have ruled out:
secondary structure:
the problem has occurred sequencing the ABI standard which shows no
secondary structure problem. Also templates will be rerun the next day,
same conditions and the problem disappears.
Template prep, primer prep:
templates from different sources fail and as above work fine the next
day.
The Gel:
We have replaced all reagents many times. No correlation. 2 gels made
at the same time by the same person loaded on different 373's. One
fast-fades, one is fine. (This happened last night after we thought we
got it).
The sharkstooth comb:
We prep this the same way. No loose acrylamide, no problems.
Sample buffer, the formamide, the blue juice:
Replaced, brand new, deionized. Still happens.
The centrisep columns:
Same lot, same technique used, one good gel, one bad gel.
The Taq enzyme, the terminator dyes:
The same lot, the same tube, one good gel, one bad gel.
The 373:
We have 2 373's. Old373 got a stretch upgrade in Apr94. New373 was
delivered as stretch Sep94. The problem has occurred on both. While it
is true that the problem has only occurred on a stretch machine, we ran
stretch gels from April to October with no problems. The problem has
occurred only once on the new 373 and has occurred 8-10 times on the
old but I feel this could easily be _not_ significant because the old
one is used more frequently.
[The thermal cycler. We have three that have run problem samples. All three check out ok]
Data collection:
The drop off does not occur at exactly the same spot (scan line) in the
gel file. It will happen in _around_ the same spot but will have enough
variation that rules out data collection problems.
Final thoughts:
Because it doesn't happen often it has been very difficult trying to
pin this down. It may have multiple reasons and they all have to be
_below_ tolerances in order for it to show up. If this is the case then
it may be a reagent or column spec. that has changed since October 94.
Has any other lab seen this problem? Anything close to this problem?
Thanks for any help or suggestions and if you know another owner of an
ABI 373, please give them a copy of this. -Paul
Paul Morrison Dana1030
Molecular Biology Core Facilities
Dana Farber Cancer Institute
1 Jimmy Fund Way
Boston, MA 02115
paul_morrison@dfci.harvard.edu
______END OF ORIGINAL POST____________________________________
___Summary_Post______________________________________________
re: ABI 373A DNA Sequencer data just fades away.(longish)
Thank you to the 14 people who responded to my sequencing problem that
I posted. Since all of them had enlightening things to say I have
included all of the responses and my original post at the end of this
synopsis.
Briefly the problem: ABI373 automated sequencing, double strand plasmid
template, cycle sequence with Taq polymerase and DYE terminators. Every
15 gels or so a dramatic loss of signal in the chromatogram in a
_group_ of samples on the gel (details below).
The reason for posting was to find out if other people had this problem
occurring in the same time frame (Oct94-present) as mine so we could
blame it on a reagent. This hasn't been ruled out but does not seem to
be the case (one response with same time frame).
Take home message from responses:
Because of the low frequency of the problem I have thought that several
factors have to fall below tolerances in order for it to occur. I now
have three favorites. Detergent formamide, and X factor.
Because it is difficult to deduce even from the raw data or the gel
file whether I have a drastic loss of fluorescence or loss of
resolution both formamide and detergent on the glass are in the running
as the culprits. For the last ten days we have deionized formamide,
capped aliquots under argon and will make new aliquots every three
weeks,(if anyone has proof this is extreme overkill please tell me). We
have also increased the rinsing procedure that follows the Alconox
cleaning of the glass plates.
The problem has not reoccurred.[it just did.] If it does I'll be back on the net to
find the X factor.
Responders:
Allison Pinder pinder@mail1.ciwemb.edu
W.Alton Jones Cell Science mbcf@transit.nyser.net
walter.just walter.just@medizin.uni-ulm.de
Mulligan, John mulligan@darwin.com
Thomas_C.Newman 22313TCN@msu.edu
Vahe Bedian DNASequence@mail.med.upenn.edu
Sheila Sheila@lenti.med.umn.edu
Tom Keller Tom_Keller@gene.biotech.wisc.edu
Mel Kronick kronicmn@ccmail.apldbio.com
Di James DI@molbiol.uct.ac.za
Steve Hardies HARDIES@thorin.uthscsa.edu
Anthony Otsuka ajotsuka@rs6000.cmp.ilstu.edu
Laura Livingstone lrl@med.unc.edu
Dave Knorr dak@biosys.apldbio.com
responses:
_____________________________________________________________________
Tom Keller Tom_Keller@gene.biotech.wisc.edu
WRITES:
RE>ABI 373A DNA Sequencer data just 2/6/95
We have seen various, intermittant, problem as well. In our case,
however,though we didn't want to believe it, it ended up being one or
more of the postcycle sequencing steps. I.e., getting the fluorescent
extension products to thewell, or a combination of these steps. The
most frequent cause is theformamide. Buying deionized formamide is not
good enough. We've done theexperiment. Freshly deionized formamide
works, month old deionized formamide,stored at 4#161# gives short
reads. We have also had a case where not payingattention to getting the
entire dried sample dissolved in the loading mixturegave intermittant
problems. Finally, we have seen samples were poor recoveryfrom the
method used to remove dye-terminators gave poor results. It isunlikely
that an instrument problem will fix itself. There's the rub.
__________________________________________________________________
Sheila Sheila@lenti.med.umn.eduWRITES:
i havent seen your intermittant problem with signals suddenly fading
out. that the problem occurs with the abi standard, and that it can be
both presentand absent on the same gel depending on the lane, makes me
think what you'reseeing is the result of something going on with the
centricep columns. do youhave two lots pooled together? or possibly
people spinning them at different speeds.by the way, we've found that
fine sephadex g50 (1 gram/16 mls ddwater) workswell in recycled
centricep plastic columns. we make up a new slurry every3 weeks. it
beats paying $2 plus dollars per column.
___________________________________________________________________
Vahe Bedian DNASequence@mail.med.upenn.edu
WRITES:
Paul,
I can't say that I have encountered the exact same problem, but let
meaddress some possibilities. We have two relatively new 373s with
stretch,and the one difficult to track problem ended up resulting from
the angle atwhich plates were held while pouring the gel. If too
verticle, hydrostaticpressure caused some separation of the plates near
the bottom. Thisresulted in lower resolution (overlapping peaks)
because the focal point ofthe laser was not exactly in the middle of
the gel. We generally had a goodstart on chromatograms, with a
generally fast but not sudden drop off ofsignal, and waves of lower-
higher resolution: ie ~60 nucl would look OK,then lower intensity and
broad peaks would appear, then it would get betteragain. Another
manifestation that correlates is variablitly in
electricalcharacteristecs and spacing. Now we clip the plates on each
side near thebottom, which does not stop the spacers from getting
thoroughly wetted, andget more consistent results. I don't know if your
problem is related tothis, but since it is an affliction of the whole
gel, I thought I'd mentionit.
Another thought that comes to mind is a malfunction of your cycler. If
itstops cycling correctly after a certain point, you would expect
exactly theresults you describe.
Good luck,
Vahe
__________________________________________________________________
Thomas_C.Newman 22313TCN@msu.edu
WRITES:
Dear Paul,
Does your problem show up on the gel image as a green smeared
background.We have been having similar problems, and in one case have
found the problemto migrate with the gel plates. ABI recently changed
vendors for the glassplates and my gut feeling is that the quality is
not to the old standard. Ifyou would like to discuss this over the
phone, you can reach me at517-353-0854. We will have both the ABI field
service engineer and the fieldapplications specialist here Tuesday (Feb
7). So if you could call me in theAM (after nine EST) I would
appreciate it or send me your phone # and I willgive you a call when I
get in. Maybe we can join forces and get some of ourproblems
addressed, if not solved. The applications specialist is coming to
"show" us how to pour a gel! Wehave only poured 3 gels a day, 5 days a
week for a year and a half...I guesswe are slow learners.
Tom Newman
DNA Sequencing Facility
Michigan State University
________________________________________________________________
John Mulligan mulligan@darwin.com
WRITES:
Paul-
We have seen something that looks something like what yo have
described. My current theory on it was that it was due to residual detergent on the glass plates (see Biotechniques Vol. 15, No. 5, p. 840). You might try other detergnets, more rinsing...
John Mulligan
Darwin Molecular Corp
mulligan@darwin.com
_________________________________________________________________
walter.just walter.just@medizin.uni-ulm.de
WRITES:
Dear Paul Morrison
you encountered severe problems with the sequencer. When I was reading
your message, I noticed that you use "blue juice".I think this may be a
problem since you may not use any dyes in theABI373a.We also had and
still have problems with the ABI. The analysis ofthe data often results
in high level peaks at the beginning of thesequence and then
immediately decreases to +/- zero. When we performa manual "CALL
BASES", we obtain all of our data. Mystery?!
Best wishes
Walter Just
________________________________________________________________
Allison Pinder pinder@mail1.ciwemb.edu
WRITES:
Paul- I am in the DNA Sequencing core facility at the Carnegie
Institution Dep't of Embryology in Baltimore. I just recieved a copy
of your ABRF memo re fast-fade gels courtesy of Betsy Nanthakumar of
Johns Hopkins. I have seenthis phenomenon crop up intermittantly since
summer of 1994. It's starting toreally bug me,too.I have only one 373
in the lab. This problem has occurred on the instrumentin its original
configuration with 24cm WTR and, since we upgraded to stretch,on 34cm
WTR gels. The same thing can occur with a wide variety of
samples:Different templates, different vectors, different
thermocyclers, prepared bydifferent people-all subject to the same
effect. Like you, I have replaced reagents and changed suppliers
several times over. The EPT doesn't indicate any electrophoresis
problems, and two ABI servicemenhave told me the machine is fine and
that I have a gel chemistry problem. Ihave changed glass plates 4
times in the past 8 months and had the sameproblem at least once with
every pair. The problem has occurred with two different software
versions. Sometimes the problem will disappear after oneof these
changes, but turn up again later. I have been at my wits' end over
this. The problem had been gone for about 2 months, then showed up
again in some samples on each of two gels last week. I am in
agreement with your thought that there may be multiple causes, all
ofwhich must be below tolerance to produce the effect. My strategy now
is turning toward getting ABI to take notice and expend some real
resources on solving the problem. Our best chance of getting them to
do this is if several people are complaining-loudly- about the same
thing. Would you let me know if you hear from others having the same
problem (I'm not on the ABRF network now)? Hope we can communicate on
solving this in the future. Thanks- Allison
Pinder
Carnegie Institution of
Washington
Department of Embryology
115 W. University Parkway
Baltimore, MD 21210
(410) 554-1207
_______________________________________________________________
Anthony Otsuka ajotsuka@rs6000.cmp.ilstu.edu
WRITES
Although we have not used fluorescence-based machines, have you
considered a problem with the amount or quality of the chain
terminators? It sounds as though the gels are working properly since
you are obtaining discrete bands. It seems as though your reactions
are terminating prematurely. This could be due to an incorrect ratio
of dideoxys to deoxys or to errors in pipetting or degraded nucleoside
triphosphates, etc. Also consider anything that could be killing the
polymerase, e.g. temperature, organic solvents, lack of magnesium, etc.
I would suggest calling the company and obtaining a good stock of
control DNA template. Most of our sequencing problems result from bad
template. At least with a uniform source of DNA you can rule out that
variable.
Good luck, Tony Otsuka
_____________________________________________________________
Mel Kronick kronicmn@ccmail.apldbio.com
WRITES:
We saw your note on the net this morning regarding gels. I can tell
you about something we have seen here that is similar, but not
identical. We have seen signals fade away and come back. As you
observed, it was difficult to correlate with other parameters. One
thing we found helped significantly was to pre-run the gels before
loading (for at least 15 minutes, preferably more like 30 minutes). We
realize that many people get away without pre-running but, as you
noted, the problem probably only occurs when several sub-optimal
conditions in the gel occur at the same time. I would be curious if my
comments have any bearing on your problem. In any event, I'll ask
around some more here before we go back East. We can discuss more when
I see you next Tuesday. Hope all else is going well.
Mel Kronick
_______________________________________________________________
Di James DI@molbiol.uct.ac.za
WRITES:
Dear Paul,
This might help. We do not have a auto sequencer, but doing manual seq.
I have noticed on occasion the same phenomena. I pinned it down to the
acrylamide. The students especially, leave traces of detergent on areas
of the plates and the matrix becomes weak or polymerisation is affected
in that area and the bands 'just fade away'.
Di James
(Sen Tech Officer)
Micobiology Sequencign Lab.
________________________________________________________________
Steve Hardies HARDIES@thorin.uthscsa.edu
WRITES:
Paul Morrison writes
[extensive description of a sporadic abberation affecting
sequence in some regions of an automated seq. gel]
My experience is limited to manual seq., but I am struck by the
similarity of your problem to the glycerol artifact. That is, the
sporadic nature (related to the way people sporadically decide more
enzyme is better), and the position and nature of the artifact (bands
spread out but not missing, and only high on the gel) sounds just like
the glycerol effect. I don't know if your sample work up removes
glycerol, but if you're not using glycerol tolerant buffer, you might
try it.
More generally, it might be anything in the samples that varies from
sample to sample, runs as a high broad band on the gel, and interacts
with the DNA running along with it. You say that the same template
will give the problem one day and not the next. I would suggest taking
such a template and doing a series where the amount of template used
varies from way too much to less than usually used. If the problem
reproducibly sets in on the high end of the template, then you're
bringing something in with the template that's causing the problem, and
the sporadicity is related to how much this substance gets concentrated
on the way to the gel. A similar strategy aimed at other components
(primers, enz) might finger the problem.
Good luck; sounds like a tough problem.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San
Antonio
Hardies@uthscsa.edu
___________________________________________________________________
Laura Livingston lrl@med.unc.edu (Laura Livingstone)
WRITES:
Hello from another Core Facility:
Have you made any progress with your problem? When you have fast-
fade, is it a resolution problem where the peaks broaden to low signal
or is the signal of the larger fragments gone or reduced? I don't
think we have had exactly the same thing but we have had major problems
with resolution quality of peaks and, like you, can "prove" that all
imaginable sources are not causing it so have been unable to track down
the source of the problem. We had alot of problems after Stretch
upgrades but both of our machines were upgraded within a month of each
other so maybe it was the upgrades just coincided with whatever the
problem is.
Laura R. Livingstone, PhD
Director
UNC-CH Automated DNA Sequencing Facility
U. North Carolina
________________________________________________________________
Dave Knorr dak@biosys.apldbio.com (Dave Knorr)
Paul:
I read your post last week about the problem(s) you are having with
your 373 runs. I'm not currently sequencing much, but your
observations sounded vaguely familiar, so I contacted a bunch of folks
here who are developing some of our sequencing chemistries.
As you pointed out there may be more than one problem going on, but the
responses I received boiled down to a couple of areas to look at.
1) Even though you replaced the formamide, it is importnat to make
sure it is truely deionized. Our QC guy runs it through resin 3X, then
freezes it in 500 microliter aliquots. He keeps those only for a
couple of weeks.
2) Yo may be having gel problems. Recently we have noticed
inconsistencies reagent mixes from a couple of suppliers. Bis-
acrylamide mixtures may not have consistent proportions of the two, and
pre-weighed pre-mixes may not have accurate weights. This will throw
off the amount of crosslinking and percentage of the gel. For in-house
QC we now use only hand-mixed gel ingredients.
3) Indeed the glycerol present in some of the reactions will cause
problems with smearing at higher molecular weights similar to what you
described. We have occasionally seen situations where gels poured
sequentially by the same person may not be consistent. Even more rare
is the situation where only a portion of the gel is good.
I hope this helps. You may want to call Technical Support at ABD about
this.
Dave Knorr
Perkin Elmer/Applied Biosystems
Agricultural Applications
dak@apldbio.com
__________END OF RESPONSES___________________________
