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Sequencing - Low Product

Sequencing - Low Product - Protocols and Methods Forum

Sequencing - Low Product - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 08-26-2005, 02:42 PM
Melody
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Default Sequencing - Low Product



Hi everyone.
My lab uses a Beckman CEQ 8000 sequencer (recently upgraded from a
2000), and it has always worked just fine, successfully sequencing
nearly all or at least quite a bit of what we are able to purify.
However, the past couple tries we have been able to sequence almost
nothing, maybe 10 out of 96 samples. The problem seems to be mainly
low sequencing PCR product. We do not usually clone, but sequence
directly from a purified PCR fragment. We used to cut bands out of an
agarose gel and clean them up with a kit but lately we have been using
Millipore 96-well plates. I am wondering if that could be the problem,
because on a gel after purifying, not all the bands show up (but out of
those that do, only a small percentage will actually sequence!).

Does anyone know of a GOOD clean-up method they can recommend for
purifying a lot of PCR fragments at a time (it would really be a pain
to cut 96 samples out of a gel)?
or
Does anyone have any suggestions about what else could be the reason we
are getting such low product when we sequence?

Here is a summary of what we have been doing:

Initial PCR at 58 C annealing temperature (this is not the problem).
Run a gel with some of the PCR to make sure we have the fragments.
Run the rest of the PCR reaction through a millipore MultiScreen PCR
96-well plate (start with 25 ul PCR rxn + 75 ul water), rinse twice
with 30 ul of water, elute in 30 ul, shake for 10 minutes, transfer to
new plate.
Run 1 ul on a gel for quantification (at this point we see less
fragments than we probably should).
Sequencing PCR on those fragments that worked up to this point - we use
the Beckman reagents but we are doing 1/4 rxns (we have been doing
those for some time and it was working before), 50 cycles of 96 C for
20 sec, 53 C for 20 seconds, 60 C for 4 min. We have also tried an
annealing temp of 55, to no avail. We use 100 ng of DNA and 10 pmol of
primer in a 10 ul reaction (do you think we are using too much
primer?).
We clean up the sequencing reaction with ethanol precipitation in the
96-well plate. We are careful, and watch to see if we lose our pellets
(since the protocol involves centrifuging the plate upside-down), but
we have lost only a few, certainly not enough to account for the more
than half that is not working, so that is not the problem.

Sorry that this was really long, but I just wanted to get as much
information as I could out there...any suggestions? Thanks in advance
for your help.

Melody

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  #2  
Old 09-02-2005, 11:14 AM
Dag Rune Gjellesvik
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Default Sequencing - Low Product

Hello,
If you have only one PCR product per reaction (no multiple bands), you can
use the SAP/Exo method. (I admit affiliation - we make the SAP enzyme).

The method removes dNTPs and PCR primers by the use of enzymes, and is well
suitable for using on multiple PCR reactions. You may obtain protocol and
product from USB or GE.

Best Regards,
Dag Rune Gjellesvik

"Melody" <[Only registered users see links. ]> skrev i melding
news:1125067339.107033.168980@f14g2000cwb.googlegr oups.com...


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