基因质粒表达到蛋白测定实验讲义.docxVIP

实验步骤篇： DNA片断纯化操作流程见图1，全套操作约需15分钟，详细说明如下。1. 向PCR反应液（或其它酶促反应液）中加入3倍量（体积）的DB Buffer（如果需加入的DB Buffer量不足100 μl时应加入100 μl），然后均匀混合。2. 将试剂盒中的Spin Column安置于Collection Tube上。3. 将上述操作1的溶液转移至Spin Column中，12,000 rpm离心1分钟，（如将滤液再加入Spin Column中离心一次，可以提高DNA的回收率。）弃滤液。4. 将500 ul的Rinse A加入Spin Column中，12,000 rpm离心30秒钟，弃滤液。5. 将700 μl的Rinse B加入Spin Column中，12,000 rpm离心30秒钟，弃滤液。（请确认Rinse B中已经加入了指定体积的100%乙醇。）6. 重复操作步骤5。7. 将Spin Column安置于Collection Tube上，12,000 rpm离心1分钟。（空甩）（对Spin Column烘干至无乙醇，一般室温10 min，烘箱50OC 5 min）8. 将Spin Column按置于新的1.5 ml的离心管上，在Spin Column膜的中央处加入25～30 μl（回溶体积）的 灭菌水或Elution Buffer，室温静置1分钟。（把灭菌水或Elution Buffer加热至60℃使用时有利于提高洗脱效率。）12,000 rpm离心1分钟洗脱DNA。（重复8但无需静置）DNA质粒提取纯化 Protocol for Purification of Plasmid DNAAdd 1.5ml overnight culture to a 1.5ml microfuge tube and centrifuge at 12,000rpm for 1 minutes. Drain the liquid completely. For low copy number of plasmid, see the protocol on the following page.（Note: the 1.5 ml microfuge can not be loaded excess bacterial, or else can not be lysed fully）Add 250ul of Solution I to the pellet, mix well，and keep for 1 minute.(Note:the bactrials must be suspended out and out, or else can not be lysed fully.)Add 250ul of Solution II to the mixture, and mix gently by inverting the tube 4-6 times and then keep at room temperature for 1 minute. To prevent contamination from genomic DNA, do not vortex.(Note:To prevent contamination from genomic DNA, do not vortex drastically. And the whole lysis time can not exceed 5 min)4. Add 350ul of Solution III, and mix gently. Incubate at room temperature for 1minute.5. Centrifuge at 12,000rpm for 10 minutes.6. Transfer the above supernatant (step 5) to the EZ-10 column. Centrifuge at 8,000rpm for 2 minutes.7. Discard the flow-through in the tube. Add 500ul of Wash Solution to the column, and centrifuge at 10,000rpm for 2 minutes.8. Repeat wash procedure in step 7.9. Discard the flow-through in the collection tube. Centrifuge at 10,000rpm for an additional 2 minute to remove any residual Wash