Deb G.
>Dear Colleagues,
>
>I am forwarding this question from a user of our genotyping facility at
>Wake Forest. Please feel free to reply directly to Dr. Yu or to the
>bulletin board. I will forward all replies to him that are posted. Thanks
>in advance for your help.
>Mark Lively
>
>>>>>>Question:
>I am writing you about the 'bleed-through' problem we are experiencing in
>our automated genotyping effort. Our genotyping is being carried out on an
>ABI 377 sequencer. We are using the CHLC/Weber genome screening set
>purchased from Research Genetics. The forward primers are labeled with
>three dyes: FAM for blue, TET for green and HEX for yellow. The problem is
>that the blue marker peaks are also present as green peaks, resulting in
>more peaks in the electropherogram. There is also a lot of noise in the
>background when the green peaks are viewed.
>Because of these problems, it is difficult to distinguish in data analysis
>between the true peaks derived from the green markers and those
>contaminating peaks from the blue markers. We have gotten contradictory
>advice ranging from using fewer PCR products when loading green markers to
>using the new dye NED in place of TET. We have found that a reliable but
>tedious method to solve the problem is to generate a new matrix regularly.
>We are wondering whether there are other people who are experiencing the
>same
>problem and whether there is a better way to solve it.
>
>Sincerely,
>Hongrun Yu, PhD
>Dept of Biochemistry
>Wake Forest Univ Sch of Med
>Winston-Salem NC 27157
>Phone: (336) 716-9057
>E-mail: yu@invader.bgsm.wfu.edu
Deborah S. Grove, Ph.D.
Manager of the Nucleic Acid Facility
Biotechnology Institute
The Pennsylvania State University
210 Wartik Lab
University Park PA 16802
http://www.biotec.psu.edu/lsc/stf/naf/naf.html