Pulsed Field Gel Electrophoresis Preparation of Chromosome-sized Yeast DNA Molecules in Solid Agarose (Modified from Schwartz and Cantor, CELL 37:67 1984 ) 1.Grow 5 ml yeast culture in YPD Broth to " stationary" (OD600 10-14) 2.Transfer 1ml to a microfuge tube ( PGC Scientific 2.2ml tube #509-220) 3.Pellet cells (20 seconds) and resuspend in 1ml pH 7.5 TE50 buffer ( 0.05 M EDTA, 0.01 M TRIS pH 7.5) 4.Wash twice with pH 7.5 TE buffer. 5.Resuspend in 0.15 ml of pH 7.5 TE50 with 2 µl zymolyase ( 20 mg/ml in 10 mM sodium phosphate pH 7.5) 6.Add 0.25 ml of 1% low melting agarose ( 1% low melting agarose in 0.125 M EDTA pH 7.5) at 42℃. 7.Immediately (<5 min) after mixing zymolyase and agar, gently mix with blue pipeete tip (3-5X) and put into CHEF disposable plug molds (Bio-Rad, 170-3713) which is cooled on ice. Zymolyase appears to become inactive upon prelonged incubation at 42 ℃. 8.Transfer plugs to a tube and add 0.4 ml LET ( 0.5 M EDTA, 0.01 M TRIS pH 7.5) 9.Incubate 8-10 hours or o/n at 37℃ 10.Transfer plugs to a 12X 75 falcon tube containing 0.4 ml NDS, pH9.5 ( 0.5M EDTA, 0.01 M TRIS, 1% N-Lauroyl sarcosine, 2mg/ml proteinase K ). Add proteinase K fresh. 11.Incubate o/n at 50℃. 12.Wash plugs 4 time in pH 7.5 TE buffer ( 1 hour each wash). 13.Store at 4℃ in 0.05M EDTA, 0.01 M TRIS pH 7.5. Program for Separating Chromosomes of Saccharomyces cerevisiae Size range: 240-2200 kb Agarose: 1.0% electrophoresis agarose Buffer: 0.5x TBE Temperature: 14℃ Switch time: 60-120 seconds Run time: 24 hours Angle: 120º Voltage gradient: 6V/cm |
→如果您认为本词条还有待完善,请 编辑词条
上一篇DNA提取及常见问题分析 下一篇基因克隆
词条内容仅供参考,如果您需要解决具体问题
(尤其在法律、医学等领域),建议您咨询相关领域专业人士。
0