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Isolation of total DNA from bacteria and yeasts by benzy lchloride method. (Zhu et al., 1993)

About 100µg of biomass from each culture transferred in clean DNA-ase free tube with 100µl benzyl buffer (100mM Tris-HCl, pH 9.0, 40mM EDTA). After shaking 20µl SDS (10%) and 50µl of benzyl chloride was added and tubes kept under 50"C for 25min, with periodically shaking on the vortex. Than 25µl, (3 M) sodium acetate saturated solution added and tubes have kept 5 min on freezer at - 20oC for precipitation of proteins. Then, after centrifugation at 6.000 rpm, 15 min the above fraction (in this stage you should see 3 layer fraction) of supernatant about 150 µl was transferred to new 1.5ml tube and 500µl isopropanol was added for DNA precipitation. Then, after centrifugation at 10.000 rpm and decantation of isopropanol the isolated DNA samples washed twice with 500µl 70% ethanol and precipitation by centrifugation at 10.000 rpm done. After decantation of ethanol precipitated DNA samples dried in vacuum evaporator at 45"C, 20min. Drying will increase DNA stability if you want to keep a long before use.

Dear Srinu

Just try the protocol given below

Single-tube LiOAc-SDS lysis (method A). Single yeast colonies (or cells collected from 100 µl of liquid YPD culture OD600=0.4) were suspended in 100 µl of 200 mM lithium acetate (LiOAc) 1% SDS solution, vortexed and incubated for at least 5 minutes at 700C (alternatively 10 minutes at room temperature). 300 µl of 96% ethanol was added for DNA precipitation, samples were mixed by brief vortexing and DNA was collected by centrifugation (15 000 g) for 3 minutes. Supernatant was removed and the pellet was washed with 500 µl of 70 % ethanol and then suspended in 100 µl of water or TE to dissolve DNA. The debris was removed by centrifugation (15 000 g; 1 minute) and 1 µl of the supernatant was used for PCR.

​ gDNA extraction by SDS treatment (method B);published in Akada et al., 2000. Single yeast colonies were suspended in 20 µl of 0.25 % SDS and vortexed for 30 seconds. Cell debris was pelleted with centrifugation and 1 µl of supernatant was used for PCR in 25 µl of total reaction volume. Triton X-100 at final concentration of 1% was added to PCR buffer.

​Glass bead beating followed by phenol-chloroform extraction (method C). Adapted from Amberg et al., 2005.Yeast cells from 1 ml of overnight YPD culture were suspended in 200 µl of lysis buffer (10 mM Tris pH 8.0, 1mM EDTA, 100 mM NaCl, 1 % SDS, 2% Triton X-100). 300 µl of glass beads and 200 µl of phenol-chloroform 1:1 was added to cell suspension. Cells were disrupted with by vortexing for 3 minutes, 200 µl of TE (10 mM Tris pH 8.0, 1 mM EDTA) wasadded and the aqueous phase was segregated by 5 minute centrifugation (approx. 15 000 g). Aqueous phase was transferred to a new tube and 900 µl of ethanol was added. DNA was pelleteted with centrifugation (approx. 15 000 g) and the pellet was washed once with 70 % ethanol. After 1 centrifugation (approx. 15 000 g) the DNA was dissolved in 100 µl of TE and 1 µl of the solution was used for PCR.

​Large scale gDNA preparation by zymolyase-SDS treatment (method D). Adapted from Sedman et al., 2005.200 ml of liquid yeast culture was pelleted by 10 minute centrifugation (approx. 2500 g). The pellet was washed with 50 ml of 10 mM Tris pH 7.5 and pelleted again by 10 centrifugation (approx. 2500 g). The pellet was then suspended in 1M sorbitol; 10mM KHPO3 pH 7.5; 1mM DTT solution and 8 mg of zymolyase was added per 1g of wet yeast pellet. The mixture was incubated for 15 minutes at 300C. Spheroplasts were pelleted by 10 minute centrifugation (approx. 2500 g) and lysed in 20 ml of 0.5M EDTA; 10 mM Tris pH 9.5; 4% SDS solution. After lysis, NaCl with final concentration 150 mM and RNase A with final concentration 10mg/ml was added. The mixture was incubated for 1 hour at 370C. Then 20 µl of Proteinase K was added and the mixture was again incubated for 2 h at 370C. After this 4 ml of phenol-chloroform 1:1 was added, the mixture is gently swirled, centrifuged for 5 minutes (approx. 2500 g) and the aqueous phase is transferred to a new tube. Then 4 ml of chloroform was added, the mixture was again gently swirled, centrifuged for 5 minutes (approx. 2500 g) and the aqueous phase was transferred to a new tube. Finally, 2 volumes of 96 % ethanol were added to the solution and DNA was pelleted by 5 minute centrifugation (approx. 5000 g). The pellet was dried and dissolved overnight in 1 ml of TE. Concentration of genomic DNA was determined by spectrophotometry.

I hope, it’ll work in your case