自制过柱法试剂盒方法.docVIP

自制过柱法质粒抽提试剂盒方法 很多人对Qiagen等公司提供的质粒抽提试剂盒很推崇，其实试剂盒的主要原理也是在碱裂解的方法上改进而成的。如果你现在还在用经典的碱裂解法提取质粒，只要去买一瓶盐酸胍（国产的纯度即可），稍作修改，就可以摆脱繁琐的酚氯仿抽提了（酚、氯仿可是有害健康的哦！！！！） 过柱法的关键原理：DNA在高盐、低PH 值条件下能吸附到含SiO2介质的纯化柱上，又可在低盐、高PH 值条件下从含SiO2介质的纯化柱上释放出来。而蛋白等杂质因为没有该种特性而不会结合到纯化柱上。 试剂配制的方法： ?I液、II液（相当于QIAGEN的Buffer P1和 Buffer P2 ）：与经典碱裂解法相同。可参考《分子克隆》中提供的配方，也可将I液中的蔗糖省略不用。 III液（相当于QIAGEN产品中的Buffer N3，关键溶液哦）：4.0M 盐酸胍，0.75M KAC，用冰醋酸将PH调整至 PH4～4.5 洗涤液（相当于QIAGEN产品中的Buffer PE）：可用70％乙醇替代。 洗脱溶液（相当于QIAGEN产品中的Buffer EB）：可用TE替代。 操作方法(借用了QIAGEN的操作步骤）： 1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. 2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. 3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy. 4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. 5. Apply the supernatants from step 4 to the QIAprep spin column（就是柱子） by decanting or pipetting. 6. Centrifuge for 30–60 s. Discard the flow-through. 7. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. 8. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. 9. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 m