[Effects of agarose and LB medium on dye-terminator DNA sequencing].
The direct sequencing of PCR products, a bacterial colony (plasmid DNA), or a phage plaque (lambda DNA) is a very powerful technique in several molecular biological applications. Recently, we reported the successful application of this direct sequencing methodology, called recyclesequencing. Occasionally, however, our sequencing efforts failed due to the presence of agarose gel containing Luria-Bertani (LB) medium. Consequently, we pursued a semiquantitative investigation of the inhibitory effects of agarose and LB medium on the direct sequencing reaction. We found that LB medium concentrations greater than 26.7% inhibited the sequencing reaction. Furthermore, agarose concentrations greater than 0.20% in a reaction mixture also inhibited the sequencing reaction.
Yamaguchi
Yukihiro
Y
Department of Biochemistry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. yyama@hyo-med.ac.jp
Nimbari
Sachiko
S
Obata
Hiroko
H
Ookawara
Tomomi
T
Eguchi
Hironobu
H
Kurotsu
Toshitsugu
T
Suzuki
Keiichiro
K
D003470:Culture Media; D012685:Sepharose; D017422:Sequence Analysis, DNA / Q000379:methods*
D003470:Culture Media; D012685:Sepharose