DNA试剂盒提取方法汉语版.doc

This method allows genomic bacterial isolation from up to 3ml LB cultureMaterials and equipment to be supplied by user:Tabletop microcentrifugeNuclease-free 1.5ml microcentrifuge tubesWater bath capable of 37Shaking water bath capable of 55Incubator or water bath capable of 65100% ethanolIsopropanolTE bufferVortexerBefore startingPrepare DNA wash buffer.HBC buffer and lysozyme as instructed in the “preparing reagents” section on page 4Set an incubaor or water bath to 65Set a water bath to 37Set a shaking water bath to 55Heat elution buffer to 651. culture bacteria in LB media to log-phase.(overnight culture can be used in many cases)2. Centrifuge no more than 3ml culture or 1109 cells at 4000g for 10 minutes at room temperature3. Aspiate and discard the media4. Add 100L TE buffer . vortex to complete resuspend the pellet5. Add 10 L lysozyme6. Incubate at 37 for 10 minutesNote: the amount of enzyme required and/or the length of incubation may need to be modified depending on the bacterial strain plete digestion of the cell wall is essential for efficient lysis.longer incubation time may yield better result.Optional:follow the short protocol belowfor difficult to lyse bacteria.1. add 25mg glass beads to 1.5ml microcentrifuge tube.2. Add sample to the glass beads3. Vortex at maximum speed for 5 minutes4. Let sample stang to allow the beads to settle5. Transfer supernatant to a new 1.5ml microcentrifuge tube.7.Add 100L BTL buffer and 20LProteinase K Solution.vortex to mix thoroughly.8.incubate at 55 in a shaking water bath.Note：usually no more than 1 hour is required for bacterial lysis.if a shaking water bath is not available,incubate the samples and shake or briefly vortex every20-30minutes.9. Add 5L RNase A.INVERT TUBE SEVERAL TIMES TO MIX10. Incubate at room temperature for 5 minutes11. Centrifuge at 10000g for 2 minutes to pellet any undigested material12. Transfer the supernatan to a new 1.5ml microcentrifuge tube.do not disturb the pellet13. Add 220L BDL buffer . votex to mix14. Incubate at 65 for 10 minutesNote:a wispy precipitate may from upon addition of BDL buffer;it does not interfere with DNA recovery15. add 220L 100% ethanol.vortex for 20 seconds at maximum speed to mix thoroughly.Note：if any precipitate can be seen at this point,break the precipitate by pipetting up ang down 10 times.16. insert a HiBind DNA Mini Column into a 2ml Collection Tube17. Transfer the entire sample to the HiBind DNA Mini Column,including any precipitate that may have formed.18. Centrifuge at 10000g for 1 minute 19. Discard the filtrate and the collection tube20. insert a HiBind DNA Mini Column into a new 2ml Collection Tube21. Add 500LHBC bufferNote:HBC buffer must be diluted with isopropanol before use .please see page 4 for instructions22. Centrifuge at 10000g for 1 minute 23. Discard the filtrate and reuse the collection tube24. Add 700L DNA wash buffer.Note:DNA Wash Buffer must be diluted with 100% ethanol before use.please see page 4 for instructions.25. Centrifuge at 10000g for 1 minute 26. Discard the filtrate and reuse the collection tube27. repeat steps 24-26 for a second DNA Wash Buffer wash step28. Centrifuge the empty HiBind DNA Mini Column at maximum speed (10000g) for 2 minutes to dry the columnNOTE:this step is criticalfor removal of trace ethanol that may interfere with downstream applications29. insert a HiBind DNA Mini Column into a new nuclease-free 1.5ml microcentrifuge tube30. Add50-100L Elution Buffer heated 65 to the HiBind DNA Mini Column NOTE: make a sure to add the elution buffer to the center of the HiBind matrix.each 50-100 Lelution typically yield 60-70%of the DNA bound to the HiBind matrix.two elutions generally yield90%. however,increasing elution volume reduces the concentration of the final product .to obtain DNA at higher concentrations ,elution can be carried out using 50L Elution buffer (which slightly reduces overall DNA yield)volumes lower than 50Lgreatly reduce yields.31. let sit for 3 to 5 minutes at room temperatureNOTE:yield may be increased by incubating the column at 65 (rather than at room temperature) .32. Centrifuge at 10000g for 1 minute to elution the DNA33. Repeat steps 30-32 for a second elution step34. Store eluted DNA at -20.该方法可从3ml LB培养物中分离细菌基因组实验者需要的材料和设备：台式微量离心机无核酸酶的1.5ml离心管(水浴锅)水浴温度37震荡的55的水浴恒温箱或65的水浴100乙醇异丙醇TE缓冲液涡流混合器实验开始之前按照第4页的“制备试剂”一节中的说明准备DNA洗涤缓冲液，HBC缓冲液和溶菌酶设置恒温器或水浴至65设置一个水浴到37摇动水浴至55加热洗脱缓冲液至65（Elution Buffer）1.将LB培养基中的细菌培养至对数期（许多情况下过夜培养可用）2.在室温下4000g离心10分钟（培养基不超过3ml或1109细胞）3.吸弃上清4.加入100L TE缓冲液。涡旋重悬5.加入10L溶菌酶6. 37水浴10分钟注意：所需的酶量和/或水浴时间可能需要根据所使用的细菌菌株进行修改。细胞壁的完全消化对于有效的裂解是必不可少的。更长的温育时间可以产生更好的结果。可：按照以下简短的方案处理难以裂解细菌。1.将25mg玻璃珠（破细胞）加入1.5ml离心管中。2.将样品加入离心管3.以最大速度涡旋5分钟4.让珠子沉淀下来5.将上清液转移到新的1.5ml离心管中。7.加入100L BTL缓冲液和20L蛋白酶K溶液涡旋混匀。8.置在55振荡水浴中。注意：细菌溶解通常不超过1小时。如果没有摇动的水浴，每20-30分钟摇动或短暂涡旋。9.加入5L RNase A。颠倒试管几次混匀10.室温5分钟11. 10000g离心2分钟，沉淀未溶解的物质12.将上清液转移到新的1.5mL 离心管中，不要搅动沉淀13.加入220L BDL缓冲液。 涡旋混合14.6510分钟注意：加入BDL缓冲液后可能会产生细小的沉淀;它不会干扰DNA的回收15.加入220L 100乙醇，最大速度涡旋混合20秒钟，使重悬充分注意：如果在这一点上可以看到任何沉淀物，可以通过反复吹打来破坏沉淀。16.将一个 DNA微柱插入2ml收集管中(2mL collection tube)17.将整个样品转移至 DNA Mini Column，包括可能形成的沉淀物。18. 10000g离心1分钟19.弃去滤液和收集管20.将 DNA Mini柱插入新的2ml收集管中21.加入500L HBC缓冲液注意：HBC缓冲液在使用前必须用异丙醇稀释，请参阅第4页的说明22. 10000g离心1分钟23.弃去滤液，依旧使用该收集管24.加入700L DNA洗涤缓冲液。(DNA wash buffer)注意：DNA Wash Buffer必须在使用前用100乙醇稀释，请参阅第4页的说明。25. 10000g离心1分钟26.弃去滤液并重复使用收集管27.对于第二次DNA洗涤缓冲液洗涤，步骤重复步骤24-2628.将空的DNA Mini Column最大速度（ 10000g）离心2分钟，使柱干燥注意：这一步对于去除可能干扰下游应用的痕量乙醇是至关重要的29.将 DNA Mini Column插入一个新的1.5ml离心管中30.加50-100L的加热到65的 Elution Buffer 到 DNA 微柱注意：确保将洗脱缓冲液添加到 matrix的中心。每次50-1