做MSP省钱法之一
丁香园论坛
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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.
~undefined*********~Kbr_~H~M~2~15._Put_the_tube_on_ice~E_add_100_uL_of_Wizard_DNA_purification_resin_~Aor_similar_DNA_purification_resin~B~E_mix~E_and_incubate_at_room_temperature_for_10_min~E_spin_at_7000g_for_30_s_at_room_temperature~E_discard_the_supernatant.~Kbr_~H~M~2~undefined*********~Kbr_~H~M~2~16._Put_1_mL_of_70~7_ethanol~E_mix_by_Vortex~E_spin_at_7000g_for_30_s~E_and_discard_the_supernatant._Do_this_two_more_times~E_and_remove_the_supernatant_completely.~Kbr_~H~M~2~17._Add_50_uL_of_TE~E_mix_well~E_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.
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.
~undefined*********~Kbr_~H~M~2~15._Put_the_tube_on_ice~E_add_100_uL_of_Wizard_DNA_purification_resin_~Aor_similar_DNA_purification_resin~B~E_mix~E_and_incubate_at_room_temperature_for_10_min~E_spin_at_7000g_for_30_s_at_room_temperature~E_discard_the_supernatant.~Kbr_~H~M~2~undefined*********~Kbr_~H~M~2~16._Put_1_mL_of_70~7_ethanol~E_mix_by_Vortex~E_spin_at_7000g_for_30_s~E_and_discard_the_supernatant._Do_this_two_more_times~E_and_remove_the_supernatant_completely.~Kbr_~H~M~2~17._Add_50_uL_of_TE~E_mix_well~E_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.
