Sharktooth Comb Leakage

Macky Edmundson mackyE at qimr.edu.au
Tue Nov 11 05:03:31 EST 1997


>Hi all
>
>We have just switched to using sharktooth combs with our Stretch/XL
>upgrade to our 373A sequencer.

I am using a 377 but have had similar problems.
>
>We are experiencing leakage round the teeth of the comb.
>
>We notice that with the "supposedly" matched spacer and comb, that after
>the gel has polymerised, the comb slides in very easily.
>
>Also, we tried using 0.3 mm spacers with the 0.4 mm comb and found that
>the comb was not tight in the middle of the gel, and we got leakage into
>surrounding slots.

3 things have improved our setup so we are gettting better gels with less
leakage:

1) Make sure the upper well is clamped BEFORE gel pouring,  pour gel then
remove clamps place gel former and reclamp, this has made a huge difference
to reproducible gel casting.

2) Wash spacers really well and make sure there is no left over acryclamide
and they are in good condition. We have had gels that run with the first
3-5 lanes off  screen, seems to be due to dirty  or creased spacers.

3) Remove "dag" acrylamide from the gel slot with a syringe/needle, squirt
1-2 mls buffer into dry well, mop up excess with a tissue, add 20-30ul of
loading buffer all the way along the slot,  place comb and then assemble
upper tank. If you put the comb in before you clamp the upper buffer tank
it is easier to fit and you get less bent/wrecked combs.

The springs  on our 377 gel caster seem to be weaker than they used to be
which is not helping . Clamping of 48cm gels using the 377 gel caster is
very difficult as the black plastic at the top of the assembly bends
especially if you try to use the adjustic screws on the white lower brace
so extra bulldog clips are needed or the comb will be really loose.

>Can kind people tell us their secrets/tips/hints/incantations to the
>gods/etc to obtaining sucessful loading of shark-tooth combs?  What is
>the leakage rate (how often do samples leak next-door?)

We had no problems with 36 well combs but sometimes get problems with 48/64
on certain gels now, obviously the quicker you can load the better.
I tend to load 64's in 3-4 steps with a 3 minute run inbetween.

>How long do people pre-run their gels ?

30 minutes usually, but can be 15 minutes if needed.

> Do you have the sharktooths
>comb in or can you insert just prior to loading?

See above , we insert combs before setting up the buffer tanks.

>Do people use the bind-silane to get smooth gel surface for inserting
>the sharktooth comb?  If so can you provide a recipe for using and how
>do you clean up after the run ?

I think silane can interefere with the laser emisson so is not recomended?

>Does anybody use the 50 and/or 66 square well former combs on a 373A XL
>upgrade?  The ABI technical help guys say the tracking isn't very good
>on the 373A XL upgrade with these combs?  Any experience out there.

Tracking is and always has been a serious problem, version 3 may be better
with 36 wells but it cannot handle blank/failed lanes and tracking of 64
lanes is pathetic.


  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 at qimr.edu.au  (Australia)
 _____________________________________________________________






More information about the Autoseq mailing list