DNA purification procedure consists of five steps and utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. During the first step cell walls of the yeast cells are disintegrated in the YS Buffer (Spheroplast Buffer). Spheroplasts obtained this way are enzymatically lysed by Proteinase K in lysis buffer. To obtain an RNA-free DNA sample, RNA is removed by RNase A. After the addition of chaotropic salts, the lysate is applied to the purification column membrane and DNA is bound. The two-step washing stage effectively removes impurities and enzyme inhibitors. Purified DNA is eluted with then use of a low ionic strength buffer (Elution Buffer) or water (pH 7.0–9.0) and may be used directly in all downstream applications such as PCR, qPCR, Southern blotting, DNA sequencing, enzymatic restriction, ligation and so forth, or stored until ready to use.
Broth or plate yeast culture, frozen cell pellet
Up to 1 x 108 cells → 100%
2 x 108 ÷ 9 x 108 → 30–70%
Approx. 40 μg DNA
Approx. 75 minutes (including incubation time)
A260/A280 ratio = 1.7 – 1.9
RECOMMENDATIONS AND IMPORTANT NOTES
DNA isolation efficiency and purity can depend not only on the number of yeast cells, cell type (morphology) or antibiotics in the grow medium, but also on the age and condition of the yeast cells.
The kit is designed to isolate DNA from max. 1 x 108 yeast cells. Using greater number of cells may significantly reduce the isolation efficiency and the purity of the DNA.
Extracting DNA from fresh starting material or frozen cell pellets is recommended. To minimize DNA degradation, avoid subjecting the sample material to repeated freeze/thaw cycles. Using old or repeatedly frozen/thawed material may result in low efficiency isolation of high molecular DNA.
EXTRACTME DNA YEAST KIT (EM10) – Discontinued - Manual
EXTRACTME DNA YEAST KIT – TROUBLESHOOTING
The quality of each production batch (LOT) of the EXTRACTME DNA YEAST KIT is tested with the use of standard QC procedures. Purified DNA concentration and quality are evaluated by gel electrophoresis and spectrophotometer.
EXTRACTME DNA YEAST KIT (EM10) – Discontinued - MSDS
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