细菌硫氧还蛋白基因组提取方法和PCR引物..docx

(一)细菌基因组的提取快速微量提取法A.取1.5ml菌体培养物于一灭菌Ep管中，12000rpm离心1min, 丢去上清夜，收集菌体。B.加入400ul裂解液（40mMTris-醋酸，20mM醋酸钠,1mMEDTA,1%SDS,pH7.8）混匀,置于37oC水浴1hr。C.然后加入200ul5mol/L的氯化钠溶液，混匀后于13000rpm离心15min。D.取上清液，用苯酚抽提2次，氯仿抽提1次。E.加两倍体积无水乙醇，1/10体积醋酸钾(3M ,pH8.0)，-20度保存1小时后，13000rpm离心15min，弃上清液，沉淀用70%乙醇洗2次；置于室温干燥后，溶于50ulTE溶液中，置4℃保存备用。蛋白酶/SDS法制备先用10ml含适当抗生素的GBM过夜培养Delftia sp.，第二天4000rpm离心10min收集菌体，用Washing TE（50mmol/LTris-HCl pH8.0,10mmol/LEDTA pH8.0）洗菌体2次，之后将菌体充分悬浮在5ml 1×TE缓冲液中，先后加入0.5ml 5mg/L的蛋白酶、0.5ml 10% SDS，轻轻混匀后50℃放置3h~5h，接着用等体积的Tris饱和苯酚抽提2次，苯酚/氯仿/异戊醇抽提一次，氯仿抽提一次后，乙醇沉淀DNA，用自动移液器吸管头将絮状DNA沉淀块吸附到Ep管中，70%乙醇洗2次，干燥后溶于适当1×TE或ddH2O中. DNA EXTRACTION PROCEDURE - GENERAL1) Grow cells overnight in 500 ml broth medium.2) Pellet cells by centrifugation, and resuspend in 5 ml 50 mM Tris (pH 8.0), 50 mM EDTA.3) Freeze cell suspension at -20C4) Add 0.5 ml 250 mM Tris (pH 8.0), 10 mg/ml lysozyme to frozen suspension, and let thaw at room temperature. When thawed, place on ice for 45 min.5) Add 1 ml 0.5% SDS, 50 mM Tris (pH 7.5), 0.4 M EDTA, 1 mg/ml proteinase K. Place in 50C water bath for 60 min.6) Extract with 6 ml Tris-equilibrated phenol and centrifuge at 10,000X g for 15 min. Transfer top layer to new tube (avoid interface). Re-do this step if necessary.7) Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting).Spool out DNA and transfer to 5 ml 50 mM Tris (pH 7.5), 1 mM EDTA, 200 g/ml RNase. Dissolve overnight by rocking at 4C.8) Extract with equal volume chloroform (mix by inverting) and centrifuge at 10,000X g for 5 min. Transfer top layer to a new tube.9) Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting).10) Spool out DNA and dissolve in 2 ml 50 mM Tris (pH 7.5), 1 mM EDTA.11) Check purity of DNA by electrophoresis and spectrophotometric analysis.碱裂解法提取细菌基因组DNA细菌DNA提取试剂盒微波法快速提取（二）引物(1 (2) CGATCCGCTGTATTTCCAAGAATTCA为EcoR1的酶切位点；CTGCAG是Pst1 的酶切位点；AAGTCC是Hind3 的酶切位点。引物设计原则与方法A.要设计引物首先要找到DNA序列的保守区。同时应预测将要扩增的片段单链是否形成二级结构。如这个区域单链能形成二级结构，就要避开它