Liz size standards

Scottie Adams sadams at aldus.northnet.org
Wed May 22 13:18:27 EST 2002

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.

>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
>>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
>>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

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.org
sadams at trudeauinstitute.org


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