Yeast DNA Extraction Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Screening of promoters and other genes of interest in the genome of S. boulardii ATCC MYA-796
Since the genome of S. boulardii is not available, we designed primers for the genes we were interested in based on the genome of S. cerevisiae s288c, available in the Saccharomyces Genome Database (http://www.yeastgenome.org/). As above, we used the ApE software to design the primers. We screened the genome of S. boulardii ATCC MYA-796 for the presence of the following promoters: PGK1 (NM_001178725.1), PYK1 (NM_001178183.1), ENO1 (NM_001181383.3) and ADH1 (NM_001183340.1). We also screened it for the replication sequences REP1 and REP2 of the episomal plasmid 2µ (J01347.1) and other genes of interest, including CAN1 (NM_001178878.1), AGA1 (NM_001183221.1), TAF10 (NM_001180474.3) and δ transposon sequence (YALWdelta1). Primers were designed to produce fragments between 100 to 300 bp, except for AGA1, which was 2,200 bp (Table 3). Both yeast species were grown overnight in 10 ml of liquid YPD and genomic DNA was extracted with Yeast DNA Extraction Kit (Pierce), following manufacturer’s instructions. As a template, we used 200 ng of genomic DNA of each yeast and performed a PCR as described above (with an elongation time of 2 min for AGA1, due to its size). PCR products were then run in a gel as described above.



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Manufacturer protocol

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