Satya and others-
OK, I will provide some details. I am using Gel Company membrane combs on both my 373XL (0.3mm thickness gel) and the 377XL (0.2mm thickness gel). We use ABI BigDye Terminators and Amersham Dyenamic ET terminators; these two kits are equally compatible with using membrane combs. We use LongRanger gel paks to prepare our gels (377-4.75% gel w/6M urea, 373-5%gel w/8.3M urea). We usually use 48cm well to read gel length but 36 cm gels on the 377 are ok for the combs too.
We did not alter our standard run conditions(volts, watts/amps) when we started using membrane combs. Those recommended by ABI and preprogrammed into the sequencing run setting for each gel length are fine. In fact, you'll find you need to make very few changes to accomodate the switch to these combs. Prepare your reactions as you always have.
Thanks to Bob Keefe for already posting a loading protocol in his 2/1/01 email. It is essentially the same as our protocol.
One difference- we use 20% Ficoll when he uses 5%. If the more dilute Ficoll works it would certainly be cheaper so I plan to try it.
Like Bob, we like the Gel Company's Rapidload trays. These are a flat piece of acrylic with tiny wells to hold samples. The wells are spaced so as to fit an 8-channel pipettor (we use Finnpipette), so we can pipette our resuspended samples from a 96-well plate directly to the rapidload tray. The trays are available in 48, 64, and 96 well options and the wells are arranged so as to allow you to dip the corresponding comb directly into them to absorb the sample liquid. If you don't have such a tray, you could lay the comb on a flat surface and pipette the samples onto the comb teeth but frankly this seems like a hassle.
Using the membrane comb eliminates the staggered loading effect we used to get with a sharkstooth comb. So, you probably will want to develop another plan to help you know that your tracked lanes are numbered correctly. Gel Company (and ABI also, I think) sell formamide loading buffer that contains a dye that runs near the front of your reactions. Load the dye in some pattern (we load it every 6th lane), then check your gel file after the run to be sure that the dye is in the right lanes. Retrack if it isn't.
Now, here's the best part if you're cheap like I am and trying to get the most out of your old equipment. (This was not my idea, but I think its a good one). If you use a 96-tooth tray and comb, you can load 96 samples on a single gel without buying ABI's 96well upgrade. Use the comb with the 1.5mm spacing, and it will fit in the original narrower plates without the carved-out trough. Set your ABI tracking software for 48 lanes. Enter two 48-lane sample sheets for your 96 samples. Of course autotracking will be useless, but with Analysis 3.3 handtracking is easy. Track and extract the first 48 lanes with the first sample sheet and store them. Then apply the second sample sheet, track the second 48 lanes, and extract and store them. This process takes about 45 minutes for us, but it allows you to do twice as many samples in one gel.
Feel free to contact me with questions. I'm sure the folks at the Gel Company 415-247-8760 would love to help you as well if that would cause you to buy more of their products! They have been very helpful to me.
Allison Pinder DNA Sequencing Core Facility
HHMI/Carnegie Institution of Washington
Department of Embryology
115 West University Parkway
Baltimore, MD 21210
(410) 554-1207
pinder@ciwemb.edu
---------- Original Message ----------------------------------
From: "Satya Yadav, Ph.D." <yadavs@ccf.org>
Date: Fri, 02 Feb 2001 09:01:09 -0500
>Dear Allison,
>
> Can you send your exact protocol for membrane combs and other run conditions such as gel %, type of gel, run voltage and who supplies membrane combs. It will help many labs who still load gels manually.
>Thanks in advance
>Satya Yadav
>
>>>> "Allison Pinder" <pinder@ciwemb.edu> 02/01/01 02:56PM >>>
>Alan- We use the membrane combs (from Gel Co.) for sequencing reactions on both the 373 and 377 without prerunning at all. We think they are great.
>
>
>Allison Pinder DNA Sequencing Core Facility
>HHMI/Carnegie Institution of Washington
>Department of Embryology
>115 West University Parkway
>Baltimore, MD 21210
>(410) 554-1207
>pinder@ciwemb.edu
>
>
>---------- Original Message ----------------------------------
>From: "Helix Biotech" <helix@execpc.com>
>Date: Wed, 31 Jan 2001 15:26:03 -0600
>
>>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.
>>
>>
>
>
>
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