质粒提取omega EZNA plasmid mini kit I

质粒提取omega EZNA plasmid mini kit I

1. 在5ml的培养基中加入5ul相应的抗生素，并用牙签在皿中挑取一个菌落，接种到该液体培养基中，摇床上 37℃，220r 12-16h扩菌。一般接种五支管。(注意标记区分，摇床上绑定一定要牢固。)

2. Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate and discard the culture media.

：16h后。每次取1.5ml的菌液于ep管中，10000rpm, 1min，在室温中。弃上清。将管倒置于吸水纸上几分钟，使培养液尽可能流尽。

3. Add 250 μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.

加入250ul的S1，吹打混匀。混匀很重要，一定要混匀（s1放在四度冰箱中） Note: RNase A must be added to Solution I before use.

Add 250 μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.

加入250ul的S2轻轻上下颠倒4-6次，出现澄清液体。静置两分钟。但是总时间不能超过5min。

Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.

4. Add 350 μL Solution III. Immediately invert several times until a flocculent white precipitate forms.

加入350ul S3，立即上下颠倒6-8次，出现白色沉淀（为蛋白质）。注意一定要立即哦 Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation. 重要的是，溶液完全混合，并立即加入后，溶液III，以避免局部沉淀。

5. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.

13000g， 10min离心.(此时可以开始配胶，准备制备管)

6. Insert a HiBind? DNA Mini Column into a 2 mL Collection Tube.

7. Transfer the cleared supernatant from Step 5 by CAREFULLY aspirating it into the HiBind? DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind? DNA Mini Column. 吸上清于制备管中，千万不要吸到沉淀。 8. Centrifuge at 10000g speed for 1 minute. 10000rpm，离心1分钟 9. Discard the filtrate and reuse the collection tube. 10. Add 500 μL HB Buffer.

11. Centrifuge at 10000g speed for 1 min

12. Discard the filtrate and reuse collection tube. 13. Add 700 μL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 80ml 100% ethanol prior to use. 14. Centrifuge at 10000g for 1 minute.

15. Discard the filtrate and reuse the collection tube.

Optional: Repeat Steps 13-15 for a second DNA Wash Buffer wash step.

16. Centrifuge the empty HiBind? DNA Mini Column for2 min at 13000g to dry the column

matrix.

Note: It is important to dry the HiBind? DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.

17. Transfer the HiBind? DNA Mini Column to a clean 1.5 mL microcentrifuge tube.

18. Add 30-50 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.

Note: The efficiency of eluting DNA from the HiBind? DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5. 19. Let sit at room temperature for 5 minute. 20. Centrifuge at 13000g for 1 minute.

Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. 21. 标记 Store DNA at -20°C. 检测鉴定： 1． 跑胶 20ml的琼脂糖胶 2ul样品 1ul的loading buffer 2. OD值

一般稀释7倍，即6ul水，1ul的样品