At 03:26 PM 1/31/2001 -0600, you wrote:
>I would like to use Gel Company membrane combs for fragment analysis on an
>ABI 373. Normally, I mount the upper buffer reservoir and pre-run the gel to
>40oC. However, with the membrane combs, they are inserted prior to mounting
>the buffer reservoir with no pre-run. Anyone have experience with membrane
>combs on the 373? Is pre-running necessary?
>
>Thanks... Alan
> Helix Biotech, Inc.
>
Alan,
I just tried some membrane combs from The Gel Co. recently (cat. no.
CAM-48) to load samples on my 373 for fragment analysis. I've only run two
gels so far, so I don't have a lot of experience to base an opinion on, but
so far, the results are pretty much the same (i.e., as good) as if I loaded
with my multi-channel Hamilton. I too usually pre-run my 373 gels to warm
them to 40 degrees, but this is not possible when using the membrane combs
for reasons outlined below. I was leary of loading/running samples without
pre-warming my gel, but the samples appeared to resolve fine just the same.
Here's a brief description of the protocol I used which was given to me by
a very helpful company sales rep located in N.C. If you work on the East
coast & want his contact info., just let me know. In fact, I reccommend
you contact him:
1. When poring gel, insert "prep" type comb at top - as if you were going
to use a sharks-tooth comb at loading.
2. Place gel/plates in 373 (do your plate check, etc.)
3. Fill bottom reservoir
4. Attach upper reservoir, but don't fill it with buffer.
5. Load 400-500 ul 5% Ficoll sol'n into prep well. I bought a 20% Ficoll
(cat. #DAL-25) sol'n from The Gel Co., and diluted it with dH2O.
6. Load samples onto membrane comb - The Gel Co. has a handy-dandy
"RapidLoad" tray for doing this ($180).
7. Insert comb through Ficoll (between plates) and place on top of
polyacrylamide, centered. If you don't center it well, you won't see all
48 lanes.
8. Quickly fill top reservoir with buffer without disturbing Ficoll
(straight-forward).
9. electrophorese 1-2 minutes (I've been doing 1.5 min) at your usual
settings.
10. stop electrophoresis.
11. Remove comb and thoroughly rinse out Ficoll sol'n from prep well (just
use buffer from top reservoir and a 60 cc syringe (no needle)).
12. Resume electrophoresis at yor usual run conditions.
I'm told the protocol changes slightly if you use the combs for sequencing.
The person to contact for more information is Wesley Moyers at The Gel
Company, tel: (336) 887-7352, email: wes@gelcompany.com. Wes was nice
enough to send me some freebie combs and a loaner on the RapidLoad tray
(which I eventually purchased). Ten CAM-48 combs cost $52. Hope this was
of some help.
Bob Keefe
Robert G. Keefe, Ph.D.
Wadsworth Center/NYS Dept. of Health
Genomics Core Facility
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