Here are some comments from some folks in the ABRF http://www.abrf.org/.
Read from the bottom up, to get the order of this.
Scottie
>>Rebecca,
>>I had noticed this when we switched to the 3700 from 373...noticed it with
>PCR products as well(not necessarily lane standard). Loading products
>segregated gives much higher signal than pooled with other PCRs. We also
>switched dye sets at the time: from fam-hex-tet-tamra (I think) to
>fam-hex-ned-rox. What dyes are in v 2.0 set? Never had the time to
>figure out what was causing it, thought maybe it had something to do with
>injection on the 3700, but if you see it in your gels, that theory gets
>deflated.
>>Lynn
>>Lynn
>>>Scottie,
>>>>I don't know if you want to pass this one on to the group, but I have seen
>>the same problem with quenching of the signal (all colors not just the size
>>standard) on 377 gels.
>>>>The interesting thing is we only see the problem when we use the ABI marker
>>set on filter D. (I never have seen it with snapshot, but then again we run
>>on 377 with no size standard.) When we run the UT marker set; 6Fam, Hex,
>>Tet, and Tamra, we have never seen the problem. This problem only came up
>>we started using the ABI marker set Version 2, the only version we have
>>used. What we found influenced the quenching the most was how much PCR
>>product was pooled in one lane. We found that we couldn't go over 2-2.5uL
>>of pooled PCR product. We found that doing an EtOH precipitation with
>>glycogen reduces the quenching effect, but does not get rid of it all
>>together. I have some rather spectacular pictures of this if anyone would
>>like to see.
>>>>When I talked with ABI about the problem the say that it is something in our
>>PCR mix since we make our own, and that we are the only customer that they
>>know of that has had this problem. But we don't think that the problem
>>comes from the PCR mix because we don't see the problem in the UT set of
>>markers. Using those Markers we load 7-10uL of pooled product.
>>>>Rebecca Scholl
>>>>--
>>Genomics Core Facility
>>University of Utah
>>30 N. 1900 E.
>>4A430 School of Medicine
>>Salt Lake City, Utah 84132
>>>>(801) 585-2977 Fax (801)585-2978
>>>>www.cores.utah.edu/genomics
>>>>>>>>>>>>>>On 5/22/02 1:56 PM, "Scottie Adams" <sadams at aldus.northnet.org> wrote:
>>>>> FYI
>>>>>> In case some of you don't monitor this group.
>>>>>>> Delivered-To: sadams at northnet.org>>>> To: autoseq at net.bio.net>>>> Newsgroups: bionet.genome.autosequencing
>>>> Date: 22 May 2002 04:37:47 +0100
>>>> From: dpboyer at wisc.edu>>>> Subject: Re: Liz size standards
>>>> Sender: owner-autoseq at hgmp.mrc.ac.uk>>>> Precedence: bulk
>>>>>>>>> We are doing Gene Scan on our ABI 3100, specifically SNaP Shot for SNP
>>>>> typing and have noticed that when we combine our Liz size standard
>>>>>with our
>>>>> reaction in HiDi formamide, the liz signal is much diminished compared to
>>>>> liz and formamide alone. What is quenching the liz signal? Anyone
>>>>>know the
>>>>> emission/excitation characteristics of Liz? Our product signals are fine.
>>>>> Thanks for the help. Please respond directly to mkeller at mail.tju.edu>>>>>>>> I actually just finished struggling against this phenomenon while
>>>> trying to create my own matrix standard (many of our customers use
>>>> filter set C which ABI doesn't support for the 3700 and 3100...but I
>>>> was successful and now I have enough for several lifetimes)... What I
>>>> finally concluded is that the reduced signal is due to competion
>>>> during the EP injection. Basicly only so many molecules can enter the
>>>> caps during the injection and the sample molecules enter in place of
>>>> some of the size standard.. I have been using 1ul of Sizestandard
>>>> plus 3ul of Sample plus 9ul of formamide and this seems to work for
>>>> all of the customer samples that I have tried in the past month (these
>>>> include: SNaP shot, AFLP, T-RFLP, and Microsats).... Now the only
>>>> caviat is that I run all of my Genescan stuff on 50cm caps using POP-6
>>>> which of course ABI does not support so if I have problems, too bad.
>>>>>>>> Hope this Helps,
>>>> Daniel Boyer
>>>> DNA Sequencing/Synthesis Facility
>>>> Biotech Center
>>>> Univ. of Wisc. Madison
>>>>>>>> ---
>>>>>>>>>>>>>>
Scottie Adams aka Pamela Scott Adams
Manager
Molecular Biology Core Facility
Trudeau Institute
100 Algonquin Avenue
Saranac Lake, NY 12983
Phone: 518-891-3080, Ext. 115
Fax: 518-891-5126
Email: sadams at northnet.orgsadams at trudeauinstitute.orghttp://www.trudeauinstitute.orghttp://www.northnet.org/mbcf
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