大家都知道,做MSP亚硫酸盐处理后,需进行纯化,我用的PROMEGE的DNA cleanup试剂盒,按它推荐的方法,每1ugDNA需纯化树脂1ml,而经过我查的文献和反复实验发现,1-2ugDNA用100ul树脂就够了,当然不再是按它的步骤,现将我的步骤公布如下,希望对大家有所帮助:
1. Take 1 -2 ug of DNA and add double-distilled water to 50 mL in a 1.5-mL tube.
2. Add 3.5 uL of 3 M NaOH (final concentration of 0.2 M), and incubate at 37°C or 10 min.
3. Freshly prepare 10 mM hydroquinone, and add 35mL of it to the tube.
4. Freshly prepare 3 M sodium bisulfite, adjust to pH 5.0 by adding 3 M NaOH, add 520 mL of it to the tube, mix well, overlay with mineral oil, and incubate at 50°C for 16 h or longer.
***********
5. Put the tube on ice, add 100 uL of Wizard DNA purification resin (or similar DNA purification resin), mix, and incubate at room temperature for 10 min, spin at 7000g for 30 s at room temperature, discard the supernatant.
***********
6. Put 1 mL of 70% ethanol, mix by Vortex, spin at 7000g for 30 s, and discard the supernatant. Do this two more times, and remove the supernatant completely.
7. Add 50 uL of TE, mix well, incubate at 50°C for 5 min, spin at 12,000g for 1 min, and transfer the supernatant to a fresh 1.5-mL tube.
8. Add 5.5 uL of 3 M NaOH, mix, and incubate at room temperature for 5 min.
9. Add 10 uL of 5 M NH4OAc and mix (for neutralization).
10. Add 1 uL of 5 M NaCl and 200 uL of 100% ethanol, mix well, keep the tube at –20°C for 1 h. Spin at 12 000g, at 4°C for 5 min, discard the supernatant, rinse the pellet with 70% ethanol, dry the pellet, and dissolve in 20–50 mL of TE, and store at –20°C.
再次声明,此法非本人原创,我只是验证而已.
扫码关注「实验菌」,入科研社群,领学习资源
更有优质直播、研选好物、福利活动等你来!