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In this study, commercial anion exchange membrane and resin were used to purify plasmid DNA. After the removal of high molecular RNA in a pretreatment procedure, various alcohol concentrations in the washing buffer were examined for purification of plasmid DNA.
In batch experiments, we found that the increase of alcohol concentrations can enhance plasmid recovery and that the buffer containing 45% isopropanol had optimal purity and recovery.
When anion exchange membrane was used for purification of plasmid DNA, calcium was added in the pretreatment to remove high molecular weight RNA. The remaining RNA and protein can be removed by using a washing buffer containing 40% isopropanol and 1.5M NaCl. The purified plasmid DNA has a purity 100% and a recovery of 43±3%.
When anion exchange resin was used for purification of plasmid DNA, 1M guanidine-Cl and 50% isopropanol were added in the pretreatment to remove high molecular weight RNA. In small scale of purification process, a washing buffer containing 1M NaCl and 40% isopropanol was used. The obtained plasmid DNA has 100% purity and a recovery close to 100%. For plasmid purification by gravity-flow, the purification condition employed buffers containing 1.5M NaCl with step-down isopropanol concentrations. The plasmid DNA of different sizes obtained by this condition had 100% purity. When the gravity-flow method was used to purify 7.5 milliliter of fermentation broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 68.5±3% and 72±5%, respectively. When purification process was scaled up to 300 milliliter broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 75±6% and 50±3%, respectively. When purification process was scaled up to 3 liter broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 70% and 20%, respectively.
In conclusion, the use of commercial anion exchange membrane and resin coupled with washing buffer of various isopropanol concentrations can be used to obtain plasmid DNA of high purity and high recovery. Additionally, the use of regenerated anion exchange resin will not affect the purity of the purified plasmid DNA.|
中文摘要 本研究係利用商業陰離子交換薄膜與樹脂純化質體核酸。藉由純化前處理將高分子量的核醣核酸移除，再藉由改變清洗液中醇類濃度探討其對分離質體核酸的影響。 在批次研究中發現，提高醇類濃度有助於質體核酸回收率的提高，且增加異丙醇濃度至45%時有最佳質體核酸回收率與純度。 以陰離子交換薄膜純化質體核酸，在利用鈣離子作純化前處理之後，以1.5M氯化鈉40%異丙醇當清洗液，可以移除核醣核酸與蛋白質，得到純度100%的質體核酸，回收率約在43±3%。 以陰離子交換樹脂純化質體核酸，則利用1.0M胍基鹽酸與50%異丙醇作純化前處理。在批次小量純化程序，利用1.0M氯化鈉與40%異丙醇當清洗液，可純化得到純度100%的質體核酸，其回收率近乎100%。而以重力流方式純化質體核酸，固定1.5M氯化鈉並以降低異丙醇濃度階梯梯度來作為純化條件，可純化不同大小質體核酸，得到純度100%質體核酸。重力流純化7.5毫升菌液時，4.7kb質體核酸與8.0kb質體核酸回收率分別為68.5±3%與72±5%。在純化300毫升菌液時，4.7kb質體核酸與8.0kb質體核酸回收率則分別為75±6%與50±3%。在純化3公升菌液時，4.7kb質體核酸與8.0kb質體核酸回收率則分別為70%與20%。 本研究利用商業薄膜與樹脂純化質體核酸，在清洗液中加入異丙醇可得到高純度高回收率質體核酸。且回收再生的陰離子交換樹脂，可以再用於純化質體核酸且不會影響其純度。
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