一简易省钱的小量质粒提取法
丁香园论坛
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这一方法简单, 省钱, 不用酚/氯仿, 可用于酶切鉴定和转染细胞测试表达情况(尤其是直接观察表达情况的荧光表达载体). 具体操作步骤如下:
Miniprep Plasmid Extraction
1. Grow bacteria O/N at 37°C with vigorous aeration in 3 ml LB or 2xYT with appropriate antibiotic.
2. Harvest by micro-centrifugation at maximum rpm (16000g) for 1 min (keep the 1 ml leftover at 4°C for making glycerol stocks).
3. Completely resuspend the bacterial pellet with 200 µl of resuspension buffer. Vortex to thoroughly resuspend the cells. Make sure the cells are completely resuspended to a homogenous suspension. Incomplete resuspension will result in poor recovery. - Resuspension (stored at 4°C): 50 mm glucose - 25 mm Tris pH 8.0 - 10 mm EDTA
4. Add 400 ul of fresh lysis buffer (prepared right before using): - Lysis buffer: 0.2 N NaOH - 1% SDS Mix by inversion for 5 x (important to be gentle). Suspension will be clear and become viscous. Leave on ice for 4 min.
5. Add 300 µl 7.5 M ammonium acetate(pH 7.8). Mix by inversion for 5 X. leave on ice for 10 min.
6. Centrifuge 15 min at 4 °C to ppt’s Xsomal DNA and Protein.
7. Transfer S/N (about 820 µl) to a new microtube (Don’t disturb the pellet).
8. Add 0.8 vol isopropanol (656 µl). Mix by inversion. Leave on RT for 10 min to precipitate DNA.
9. Centrifuge for 10 min.
10. Aspirate S/N, wash pellet 2X with 500 µl of cold 70% EtOH (Prepared from molecular grade 100% EtOH)
11. Dry pellet at RT for 10 min. (Don’t over dry DNA pellet. Over-dried DNA pellet can’t be dissolved in buffer or ddH2O)
12. Dissolve pellet in 30 µl ddH2O containing 100 µg/ml RNase (Stock 10mg/ml)
13. Use 0.5~1.0 µl DNA sample for restriction analysis
14. Select bacterial strains with a positive restriction digestion result. Make 10% glycerol bacterial stocks (200 µl of 50% glycerol + 800 µl bacterial culture). Store at – 80°C
Miniprep Plasmid Extraction
1. Grow bacteria O/N at 37°C with vigorous aeration in 3 ml LB or 2xYT with appropriate antibiotic.
2. Harvest by micro-centrifugation at maximum rpm (16000g) for 1 min (keep the 1 ml leftover at 4°C for making glycerol stocks).
3. Completely resuspend the bacterial pellet with 200 µl of resuspension buffer. Vortex to thoroughly resuspend the cells. Make sure the cells are completely resuspended to a homogenous suspension. Incomplete resuspension will result in poor recovery. - Resuspension (stored at 4°C): 50 mm glucose - 25 mm Tris pH 8.0 - 10 mm EDTA
4. Add 400 ul of fresh lysis buffer (prepared right before using): - Lysis buffer: 0.2 N NaOH - 1% SDS Mix by inversion for 5 x (important to be gentle). Suspension will be clear and become viscous. Leave on ice for 4 min.
5. Add 300 µl 7.5 M ammonium acetate(pH 7.8). Mix by inversion for 5 X. leave on ice for 10 min.
6. Centrifuge 15 min at 4 °C to ppt’s Xsomal DNA and Protein.
7. Transfer S/N (about 820 µl) to a new microtube (Don’t disturb the pellet).
8. Add 0.8 vol isopropanol (656 µl). Mix by inversion. Leave on RT for 10 min to precipitate DNA.
9. Centrifuge for 10 min.
10. Aspirate S/N, wash pellet 2X with 500 µl of cold 70% EtOH (Prepared from molecular grade 100% EtOH)
11. Dry pellet at RT for 10 min. (Don’t over dry DNA pellet. Over-dried DNA pellet can’t be dissolved in buffer or ddH2O)
12. Dissolve pellet in 30 µl ddH2O containing 100 µg/ml RNase (Stock 10mg/ml)
13. Use 0.5~1.0 µl DNA sample for restriction analysis
14. Select bacterial strains with a positive restriction digestion result. Make 10% glycerol bacterial stocks (200 µl of 50% glycerol + 800 µl bacterial culture). Store at – 80°C
