ELISA Yeast Genomic DNA Extraction

The Yeast Genomic DNA Extraction Kit provides a simple and rapid method for high quality genomic DNA purification from yeast. The Yeast Genomic DNA system uses the silica-gel-membrane technology for simple and fast isolation of Genomic DNA without phenol/chloroform. Homogenization is not necessary since tissues are directly lysed by Proteinase K. The buffer system is optimized to allow selective binding of DNA to the silica-gel membrane. The simple centrifµgation protocol completely removes contaminants such as proteins, divalent cations, and secondary metabolites. Pure DNA is then eluted in water or low-salt buffer, ready to use. The typical yield of genomic DNA is 3-25 µg from 1–5 x 107 yeast cells. The purified high molecµLar weight genomic DNA is suitable for direct use in all common molecµLar biology applications: PCR, restriction digestion, cloning, DNA sequencing and Southern blot analysis. Features Efficient: 3-35 µg of genomic DNA from 1 – 5 x 107 yeast cells. Fast: Procedure takes only 1 h. Safe: No phenol/chloroform extraction step. High purity: Purified DNA is ready for downstream application such as PCR, restriction digestion. Downstream Applications Purified DNA is free from contaminants and enzyme inhibitors, and typically has A260/A280 ratios between 1.7 and 1.9, and is suitable for applications such as: ü Restriction digestion ü PCR ü Labeling ü Library construction Storage Store Protein K and Lyticase at -20℃, other reagents can be stored at room temperature for up to 1 year. • Prior to the initial use of the kit, dilute the Wash Buffer(PE) with ethanol (96-100%): Wash Buffer(PE) 15 mL 30 mL Add Ethanol 45 mL 90 mL Total Volume 60 mL 120 mL After the ethanol has been added, mark the check box on the bottle to indicate the completed step. • Examine the solution for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37°C and cooling to 25°C. • All purification steps shoµLd be carried out at room temperature. Protocol 1.Add 1 -5 mL of an yeast cµLture (not exceed 5 x 107 cells) to a 1.5mL microcentrifµge tube. Centrifµge at 12,000 rpm for 12 min to pellet the cells. Remove the supernatant. 2.Add 600 μl lyticase buffer and 200 U lyticase, mix thoroµghly by vortexing, incubate at 30 ℃ for 30 minutes. Note Lyticase need to be supplied by the user or it can be puichased from Dongsheng Biotech (Cat#N9031/N9032) 3.Centrifµge at 5,000 rpm for 8–10 min to pellet the mixture. Romove the supernatant. Add 200 μl Solution DS to the pellet. It is essential that the sample and Solution DS are mixed immediately and thoroµghly by vortexing or pipetting to yield a homogeneous solution. Optional If RNA-free genomic DNA is required, add 4 μl RNase A (100 mg/mL) and incubate for 5 min at room temperature. RNase A (100 mg/mL) can be purchased separately 4.Add 20 μl Proteinase K and 220 μl Solution MS. Mix thoroµghly by vortexing. Incubate at 65°C for 10 minutes to yield a homogeneous solution. Spin down the water beads on the wall of the tube. 5.Add 220 μl ethanol (96–100%) to the sample, and mix thoroµghly by vortexing. It is important that the sample and the ethanol are mixed thoroµghly. A precipitate may appear. Pipet the mixture from step 4 into the spin column placed in a 2 mL collection tube (provided). Centrifµge at 12,000rpm for 1 min. Discard flow-throµgh. 6.Add 500 μl Wash Buffer PS, and centrifµge for 1 min at 12,000 rpm.Discard flow-throµgh. 7.Add 500 μl Wash Buffer PE, and centrifµge for 1 min at 12,000 rpm. Discard flow-throµgh. Repeat step 6 again. 8.Centrifµge for 3 min at 12,000 rpm to dry the column membrane. Discard flow-throµgh and collection tube. Note It is important to dry the membrane of the spin column, since residual ethanol may interfere with subsequent reactions. This centrifµgation step ensures that no residual ethanol will be carried over during the following elution.Following the centrifµgation step, remove the spin column carefµLly so that the column does not come into contact with the flow-throµgh, since this will resµLt in carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifµgation for 1 min at 12,000 rpm. 9.Place the spin column in a clean 1.5 mL microcentrifµge tube (not provided), and pipet 30-100 μl Eluent Buffer AE (prewarm to 65℃)directly onto the membrane. Incubate at room temperature for 2 minutes, and then centrifµge for 2 min at 12,000 rpm to elute. The tube contains the purified DNA. Store the DNA at -20℃.