Dear fellow ABI sequencers,
Thanks ever so much to everyone who contributed their thoughts and
advice regarding the question of gel extrusion (I accidentally called
it gel creeping in my original posting in a Norwegian-English direct
translation!).
I've included a long summary, because there may be several factors
playing a role, and you may benefit from reading all of the replies, if
you're having problems.
Good luck!
Andrew
Short Summary
-------------
As charge builds up on the plates, the gel may extrude during a run -
sometimes fatally to the run. There is no clear, conclusive method for
eradicating the problem, based on the information given by users,
outlined below. It does appear, however, that the water used to
wash/rinse the plates must be as pure as pure can be. Another theme is
that of a dishwasher - perhaps through the high temperature for
washing, and particularly drying. We're testing out a dishwasher now.
Our lab have also found that the problem is worse on hot humid days
(despite the air conditioning), and that Long Ranger appears to extrude
less frequently and much less severely. This has not been tested
scientifically. We should probably also follow PE's advice (see below)
which may help to an extent, in a prophylactic way.
Personal Addition
-----------------
Having heard so many plate cleaning protocols, I thought I'd add: We
lower a dry paper towel directly onto the gel after a run, and
lift/roll it off gently. This removes the whole gel in one go, and
elimnates the need to use detergents - the plates can be washed in
water only.
Summary of Individual Replies
-----------------------------
These are anonymous, because it would take too long to write to each
perwson to ask permission to use names! If wyou want to contact a
person about their experience, e-mail me, and I'll let them know.
>From summary of replies to the same question last year to this newgroup:
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Wash new plates with Alconox & rinse well. Dry plates in 30°C oven
overnight.
Reply 1
+++++++
Solve gel extrusion: Soak the plates in warm Alconox and Dawn solution
for a few hours.
Normal handling: Wash plates with Alconox/Dawn mix & household sponge.
Air dry plates then use ethanol. Never let plates sit with the gel in
them. Always rewash plates before use.
Suggestion: Try changing TBE. Uses Amresco TBE sold in pack of 2 foil
pouches, each pouch making 1 L 10X TBE.
Reply 2
+++++++
Solved gel extrusion: Wash plates with hot water & Alconox. Rinse well
with tap water. Rinse with DI water. Wipe the gel side of the plate
vigorously with a HPLC grade methanol soaked Kimwipes (Kleenex
tissues). Rinse plates again with DI water. Final rinse from squeeze
bottle with ultra pure water. Air dry plates in rack.
Suggestion: Etch plates to ensure correct orientation.
Reply 3
+++++++
Normal Handling: Charge builds up on plates. Acid wash to counter
this. Use Alconox or Microcleaner to remove gel build-up. They
hand-wash plates with hot tap water and dilute Microcleaner soap.
Rinse plates thoroughly with hot tap water, rinse again with deionised
water not RO water but completely de-ionised. Once gel starts
pinching, especially Genescans, they do an acid wash. Don¹t do this
too often, as acid builds up -> other problems.
Reply 4
+++++++
Suggestion: Traced the problem back to a bad (old) batch of TRIZMA in
the TBE buffer. His impression at the time was that the problem was
worse on hot humid days.
Normal Handling: "We seem to be the only lab in the world that never
washes gel plates they are taken apart promptly in the morning
following the run, rinsed with hot tap water while being scraped with a
razor blade as necessary, rinsed again with distilled water, and
finally 100% EtOH and air dried. We never use Alconox or any other
detergent. We have used the same plate pair for several years with no
problems."
Reply 5
+++++++
Suggestion: "Do not use tap water for washing the plates, this is
probably the cause of your problems. Use only distilled/ deionised
water". Rinse with HCl or HNO3 now that they have become contaminated
with ionised particles. Alcoholic NaOH will also work.
Further comments: We only wash with cold dd water.
Reply 6
+++++++
Suggestion: Wash plates with KOH or HCl at least once a month, else
hard to pour. "It appears there is a gradual build-up of a film on the
plates that can cause problems like the infamous red rain. This is
solved by the wash, but can cause gel extrusion as a result. "The
reason is that the plates have a charge that attracts the acrylamide to
them. This can be stripped if the plates are not fully neutralised,
hence the HCl treatment after KOH".
Solved gel extrusion: Soaked plates overnight then rubbing them with a
cloth or sponge under running water. This appears to restore the
charge, and the problem went away.
Reply 7
+++++++
Solved gel extrusion: Washed plates with NaOH.
Normal Handling: Previously water/Alconox then NaOH and HCl. "We now
use a glassware washer and always have very clean backgrounds, and no
gel extrusion".
Reply 8
+++++++
Water purity is of utmost importance. Experienced extrusion when water
purification system was dysfunctional.
Perkin Elmer: Genetic Analysis FAQ
++++++++++++++++++++++++++++++++++
<http://www2.perkin-elmer.com/ab/techsupp/tra2b5.html>
[Quote]
"What is the best way to keep my plates clean?"
Less is best. Use a mild detergent, such as "Alconox", for daily
cleaning. A liquid solution such as "Liquinox" or even "Alconox" in
solution is an alternative for daily cleaning. You would still want to
use the powder from time to time to get any bits of acrylamide that may
be sticking to your plates. Over time there may be a buildup of
acrylamide. If you notice a problem with gel extrusion or even visible
buildup, you may want to try the following:
Use saturated alcoholic KOH solution. This is approximately 3% KOH in
isopropanol or ethanol, be sure that you add enough pellets so that it
won¹t all go into solution, then you know it is saturated. You would
want to soak your plates for 2-5 minutes and then rinse them very well
with distilled water. This solution is very corrosive and will etch the
plates if they soak too long. If you still have buildup, try the
treatment again.
***CAUTION: Be sure to wear protective clothing for this, it¹s really
nasty stuff!
You could also try a 3M NaOH or 3M HCl. These solutions may work for
you but neither of them will work as well as the alcoholic KOH.
[Unquote]
--
Institute of Immunology (IMMI)
The National Hospital, N-0027 Oslo, Norway
Tel: (+47) 22868703 (office) or 22868704 (lab)
http://www.brunel.ac.uk/depts/bl/project/biocomp/sequence/seqanal_guide/
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