Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers.
Yeast is widely used in molecular biology. Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants. We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature.
Haaning
J
J
Department of Molecular and Structural Biology, University of Aarhus, Denmark. jh@mbio.aau.dk
Oxvig
C
C
Overgaard
M T
MT
Sottrup-Jensen
L
L
D001483:Base Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis*; D003902:Detergents; D006358:Hot Temperature; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic
D017931:DNA Primers; D004271:DNA, Fungal; D003902:Detergents