The amplified DNA was subjected to gel electrophoresis using
equipment not previously used for other yeast DNA samples.
Freshly prepared gel buffers were used, and the gel cast apparatus
was previously treated with DNA-Zap to eliminate contamination
from modern DNA or RNA. DNA blank extractions, serving as
negative controls, were carried out in parallel during the extraction
and the PCR amplification. A positive control was also carried out
by adding 50 ng of modern S. cerevisiae genomic DNA to an aliquot
of the negative control prior to electrophoresis. The amplifications
were performed using Hi-Fidelity DNA polymerase
(Hoffmann–La Roche, Basel, Switzerland). The amplification reaction
conditions were as described by Guillamo´ n et al. (1998). The
DNA amplifications were carried out twice at different times and in
different laboratories and resulted in products that were indistinguishable
from one sample to the next. After extraction of bands of
the amplified DNA from the gel, DNA sequencing was carried out
directly on the PCR products without prior cloning, employing the
same primers as used for amplification and internal primers
as needed. The putative amplification product from ancient
S. cerevisiae was sequenced from samples from two different
extractions.
The amplified DNA was subjected to gel electrophoresis using
equipment not previously used for other yeast DNA samples.
Freshly prepared gel buffers were used, and the gel cast apparatus
was previously treated with DNA-Zap to eliminate contamination
from modern DNA or RNA. DNA blank extractions, serving as
negative controls, were carried out in parallel during the extraction
and the PCR amplification. A positive control was also carried out
by adding 50 ng of modern S. cerevisiae genomic DNA to an aliquot
of the negative control prior to electrophoresis. The amplifications
were performed using Hi-Fidelity DNA polymerase
(Hoffmann–La Roche, Basel, Switzerland). The amplification reaction
conditions were as described by Guillamo´ n et al. (1998). The
DNA amplifications were carried out twice at different times and in
different laboratories and resulted in products that were indistinguishable
from one sample to the next. After extraction of bands of
the amplified DNA from the gel, DNA sequencing was carried out
directly on the PCR products without prior cloning, employing the
same primers as used for amplification and internal primers
as needed. The putative amplification product from ancient
S. cerevisiae was sequenced from samples from two different
extractions.
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