We dilute our primers to 3.2 pmol/ul in water, and then use 1 ul per
reaction. I don't know how much TEA or Amm acetate salts would be present in
this small amount, but it doesn't seem likely to cause trouble. Sequencing
primers should have a melting temperature of at least 50 degrees C. The
background might come from non-specific binding of the primer.
Let me guess: Bad sequencing results are YOUR fault, right?
Sheryl
OMRF DNA Sequencing Facility
Oklahoma City, OK
www.omrf.ouhsc.edu
sherylc@omrf.ouhsc.edu
Alain Laurent wrote:
> Hello ABRF's users
>
> We recently had a complaint about HPLC purified oligonucleotides used for
> sequencing. The hybridization versus the target seems OK but there is a
> strong noise background on the electrophoregram making the reading
> difficult. Do you think that triethylammonium or ammonium acetate salts
> used for HPLC purification
> could be responsible for this effect ? I don't know if this can be a
> plausible explanation for unsuccessful sequencing ?
> By the way do you know the main causes for bad results in sequencing ?
>
> Thank You
>
> **************************
>
> Alain LAURENT Ph. D
> Oligonucleotide Chemistry
> ESGS Groupe CYBERGENE
> 11 rue Claude Bernard
> 35400 SAINT MALO
> FRANCE
> Tel: +33 (0)2 99 21 90 40
> Fax: +33 (0)2 99 21 90 41
> e-mail: a.laurent@eurosequence.com
> http://www.eurosequence.com
>
> ***************************