Fellow ABRF board members,
A colleague here at Wake Forest would like to ask the following
question about genotyping. We would appreciate your input. Please feel
free to post your reply to the ABRF BBS or to contact her directly.
Mark Lively
> Goal: To genotype a 46bp variable length tandem repeat, that exhibits
> 10 alleles ranging in size from approximately 450 bp (this includes
> 262 bp of flanking sequence) to about 1100 bp.
>
> Conditions: The following conditions have been suggested; 6-FAM
> labeled primer, GS-2500 TAMRA size standard, run on a non-denaturing
> 5% PAG, using either matrix A or D on the ABI 377 DNA sequencer for
> approximately 10 hours. While these are suggestions, I have not been
> able to identify anyone who has used these conditions to successfully
> genotype large fragments across such a broad range (450-1100bp).
>
> Question: Does anyone have experience in genotyping fragments of this
> size? What conditions can they recommend (non-denaturing vs.
> denaturing, length (time) of run), appropriate matrix) ? Are there
> specific conditions to avoid?
>
> Thank you for your input.
>
> Jeannette Bensen
> jbensen@wfubmc.edu
-- Mark O. Lively, Ph.D. Professor of Biochemistry Wake Forest University School of Medicine Medical Center Blvd. Winston-Salem, North Carolina 27157 Voice: 336-716-2969 Fax: 336-716-7200 email: mlively@wfubmc.edu
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