>
>I've suggested the cause is excess salt in the samples, resulting in
>increased conductivity
>within the wells of that set of samples, and so skewing the electric field
>and hence the lane
>pattern.
>
>Is that sensible? Does anyone have a better idea? What can be done to
>eliminate the
>problem?
Definately sounds like salt in reactions, usually caused by poor ethanol
precipitation.
I usually solve the problem by taking the users responsible outside and
shooting them,
alternatively you can try to educate them but this is a lot harder.
We have not had any problems with skewing caused by formamide or acrylic
acid, but I have had occasional gels that have had 3-4 lanes skew off the
scan region. This seems to have been due to the spacers on one side being
dirty hence slightly thicker.
Here is the protocol I give to new users:
ETHANOL PRECIPITATION
Leftover salt from a poor precipitation will cause lane compression of your
sample as well as tracking errors and can cause severe disruptions to the
entire gels.
If you continually have samples with bad dye blobs/salt you may be asked to
stop using the service until you can provide samples of a sufficently high
standard .
We recommend the following modified Ethanol Precipitation Protocol;
1) add 2.0ul 3M sodium acetate (pH4.6) and 50ul 95% Ethanol to a 1.5 ml
eppendorf. Transfer your reaction mix ( 20ul full Reaction) to a NEW
eppendorf, vortex and place on ice for exactly 10 minutes if using Big Dye
and ~30 minutes for dRhod.
USE AS LITTLE OIL AS POSSIBLE in your PCR's. There should be no oil in you
sequencing samples as it interferes with gel loading and is often
associated with salt and dye blobs. Excess oil can be removed by placing
your reaction mix on a piece of parafilm then pipetting off the reaction
mix leaving the oil behind.
2) Centrifuge for 20 minutes at max speed.
3) Remove all leftover liquid ,you can use an aspirator, a pipette or
simply decant
as long as you are rigorous. It is usually easier to decant most of the
liquid, respin for 10-20 sec then remove the remaining liquid with a
pippetor. There should be no visible liquid leftover and be careful
as the DNA pellet can easily be dislodged. Try to use a pippette tip that
has a very fine or bevelled point (e.g. gel loading tips), especially for
removing the last droplets .
4 ) Rinse the pellet with 500 ul 70% ethanol, mix then re-spin for 15
minutes. Do not leave pellet in ethanol for long periods. The pellet is
usually very small or may not even be visible.
5) Once again remove all liquid as completely as possible! Dry your samples
under vacuum or in a speedy-vac. Please use only 1.5ml Eppendorfs.
Unreacted Big Dye terminator seems to be harder to remove than previous
dye types this may be due to co-precipitation of dye with the DNA. To
reduce this please do not leave sample to precipitate for more than
15minutes.
dRhod samples actually do not precipitate as easily so longer incubation
and spin times are recomended.
Macky Edmundson - Queensland Institute of Medical Research
Socrates - To Be is To Do
Jean Paul Sartre - To Do is To Be
Frank Sinatra - Do Be Do Be Do
email mackyE@qimr.edu.au (Australia)
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