A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells.
A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.
Klebanow
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ER
Department of Molecular Physiology & Biophysic, Vanderbilt University, School of Medicine, Nashville, TN 37232-0615, USA.
Weil
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PA
D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D015698:Genomic Library; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D013104:Spheroplasts / Q000166:cytology / Q000235:genetics; D013698:Templates, Genetic; D013997:Time Factors; D015003:Yeasts / Q000166:cytology / Q000235:genetics* / Q000254:growth & development
D005656:Fungal Proteins