PLASMID DNA ISOLATION-ALKALINE LYSIS METHOD

1)  Add 3ml of LB medium and 30 ul of ampicilin (10mg/ml) to a culture tube, inoculate the tube with a single isolated white colony picked from an LB-Ampicillin plate. Incubate in the shaker (37℃, 225 rpm) for 12-16 hrs.

2)  Prewarm 3 solutions. Centrifuge 1.5mls of the culture in a 1.5ml tube for 1 min at 6,000g (check the speed of the centrifuge). Discard the supernatant. Resuspend the bacterial pellet in 200ul of Solution I. Mix well by scraping on a rack back and forth for 10 times. 

3)  Add 400ul of Solution II. Mix by inverting the tube gently for 10 times. Let sit at RT for less than 30s min until the solution is clear. 

4)  Add 300ul of Solution III. Mix by inverting the tube gently for 2 times then with the tube inverted flip the bottom of tube. Centrifuge the tube for 5 min at 18,000g. Transfer the supernatant (about 800ul) to a fresh tube.

5)  Add 1ul of boiled RNase A(25mg/ml), invert 5 times, incubate at 65 centi-degree for 5 min.

6)      Wear new gloves. Add 500ul of PCI, vortex for 30 sec. Spin for 3min, Take tube out of centrifuge gently. Transfer the supernatant to a new tube.

7)      Add 500ul of chloroform, vortex for 30 sec, spin for 3 min. Transfer the supernatant to new tube.

8)      Add 800ul-1ml of 100%ETOH to the supernatant. Mix well by inverting the tube several times. Let sit at RT for 2min, centrifuge at 18000g for 5min. White pellet should be seen. Remove and discard supernatant.  Add 800ul-1ml of ice cold 75%ETOH. Break the pellet by pipette tip ( it should be broken easily), centrifuge again for 5min.  Remove and discard supernatant by pipetting. Air dry for about 30min-1h. It should become transparent.

9)      Add 50ul of sterile ddH2O to the tube, let sit at RT for 30min to dissolve it.  Nanodrop it to check the concentration, peak, 260/280, 260/230, and run 1-2 ul on gel to see the quality.

10) Save at -20 degree in a tube.

It takes about 2.5 hrs to complete the extraction.

BUFFERS

Solution I:  50 mM glucose, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA. 

For 100ml:  Add 0.9g dextrose, 5ml of Tris-HCl (PH8.0), 2ml of 0.5M EDTA (PH8.0) to 80ml of MilliQ H2O. Adjust the volume to 100ml with MilliQ H2O.Keep at 4^oC to prevent growth of contaminants.

Solution II  200mM NaOH, 1%SDS

For 10ml: 0.2ml 10N NaOH, 1ml 10% SDS, 8.8ml dH2O.

Solution III  3 M potassium/5 M acetate, pH5.5 

For 100 mL, take 29.4 g of potassium acetate, add water to 88.5 mL, and 11.5 mL of glacial acetic acid. Store at room temperature.

说明：

1. 手工抽提与试剂盒抽提相比，优点是质量高（260/280, 260/230比值更高），缺点是麻烦，耗时多，且要接触PCI。Kit一般四五十分钟，手工抽提要2.5个小时；配PCI很麻烦，且只能保质几个月。

2. Buffer I 和Buffer III一年内有效，Buffer II配好后两个月内有效，失效的原因一般是NaOH吸收空气中的CO2。

3. 常见问题：（1）提不出质粒或者产量低：大肠杆菌老化，杂菌污染，碱裂解不充分,或者solution量不够，蛋白质沉淀不充分，导致DNA酶残留消化了质粒；（2）耐受酶切：任何物质残留都可能导致耐受酶切。例如蛋白质，多糖，乙醇，酚，盐类等。PCI氧化失效、碱裂解时间过久也可能导致耐受酶切。此外，变性超螺旋质粒会耐受酶切（导致酶切不完全）。（3）部分耐受酶切：因为有变性超螺旋的存在，可能是碱裂解时间过长，超螺旋质粒双链解开，同时由于操作不轻柔，感染了复性，配对错误，酶切位点丢失。防止方法：沉淀细菌的离心力不要太大，否则难以resuspension，4000-6000g惟一。充分重悬浮细菌，不仅要看不到贴在tube底部的菌块，还要保证悬浮后为单个细菌而不是小的细菌团。充分悬浮，可以缩短裂解时间。加入solution II之后，30秒之内要变澄清。加入solution III，操作要非常轻柔。总的来说，要点就是：充分悬浮，快速裂解，操作轻柔。（3）质粒降解：常常是由于solution量不够，培养液过多，省略了PCI抽提，导致DNA酶残留。在-20度保存于蒸馏水的情况下，降解不明显，一旦酶切后，因为有了buffer，DNA酶变得有活性，降解非常明显，甚至在-20度也快速降解。

4. 在乙醇沉淀后，沉淀物应该为白色粘稠物，用tip可以打碎，不如基因组DNA粘稠，干燥后透明。如果干燥后不透明，可能是蛋白质或者盐。

5. 尝试过一次不用PCI抽提，3毫升培养液，加入3倍体积的solution，结果费时一样多，且导致DNA酶残留，酶切后降解很严重。