【交流】核酸酶P1酶解和牛小肠碱性磷酸酶配置和注意事项
丁香园论坛
2226
1 溶解核酸酶P1（sigma)的冷冻干燥物与1.0ml30mM醋酸缓冲液（PH=5.3）？不知道此配法行不行？在sigma的说明书上没有具体说明！
酶活性:1mg核酸酶P1在1小时，ph5.3 ,T=50 能够把2gRNA或0.2gDNA酶解为5-单核甘酸
促进此酶的稳定性：在孵育时，可加zn和人血清白蛋白增加其稳定性！
2牛小肠碱性磷酸酶
试剂准备
• 10mM Tris-HCl (pH 8.0)
• CIAP stop buffer
10mM Tris-HCl (pH 7.5)
1mM EDTA (pH 7.5)
200mM NaCl
0.5% SDS
• TE-saturated phenol:chloroform
Mix equal parts of TE buffer and phenol and allow the phases to separate. Then mix
1 part of the lower, phenol phase with 1 part of chloroform:isoamyl alcohol (24:1).
• chloroform:isoamyl alcohol (24:1)
• 7.5M ammonium acetate (pH 5.5)
• ethanol, 100% and 70%
具体步骤：酶解5-去磷酸化
1. Dilute sufficient CIAP for immediate use in CIAP 1X Reaction Buffer to a final
concentration of 0.01u/μl. Each picomole of DNA ends will require 0.01u CIAP.
(1μg of 1,000bp DNA = 1.52pmol DNA = 3.03pmol of ends.)
2. Purify the DNA to be dephosphorylated by ethanol precipitation, and resuspend
the pellet in 40μl of 10mM Tris-HCl (pH 8.0). Set up the following reaction:
DNA (up to 10 pmol of 5´-ends) 40μl
CIAP 10X Reaction Buffer 5μl
Diluted CIAP (0.01u/μl) up to 5μl
50μl
3. Incubate at 37°C for 30 minutes.
4. Add another aliquot of diluted CIAP (equivalent to the amount used in Step 2), and
continue incubation at 37°C for an additional 30 minutes.
5. Add 300μl of CIAP stop buffer. Phenol:chloroform extract and ethanol precipitate
by adding 0.5 volume 7.5M ammonium acetate (pH 5.5) and 2 volumes of 100%
ethanol to the final aqueous phase.
[b]Note: CIAP may be added directly to digested DNA. Add 5μl CIAP 10XReaction Buffer, 0.01u CIAP/pmol of ends and deionized water to a finalvolume of 50μl (6).
有没有哪位战友配过这两种试剂，能否将经验一起分享一下！因为第一次做相关试验没有经验！谢谢！
eg:1 配好了液体并保存在冰箱里，酶的稳定性如何，能存多久？特别是核酸酶P1
2 酶解组织DNA的反应体系如何确定？具体操作如何？因为查相关文献具体步骤较简单！
酶活性:1mg核酸酶P1在1小时，ph5.3 ,T=50 能够把2gRNA或0.2gDNA酶解为5-单核甘酸
促进此酶的稳定性：在孵育时，可加zn和人血清白蛋白增加其稳定性！
2牛小肠碱性磷酸酶
试剂准备
• 10mM Tris-HCl (pH 8.0)
• CIAP stop buffer
10mM Tris-HCl (pH 7.5)
1mM EDTA (pH 7.5)
200mM NaCl
0.5% SDS
• TE-saturated phenol:chloroform
Mix equal parts of TE buffer and phenol and allow the phases to separate. Then mix
1 part of the lower, phenol phase with 1 part of chloroform:isoamyl alcohol (24:1).
• chloroform:isoamyl alcohol (24:1)
• 7.5M ammonium acetate (pH 5.5)
• ethanol, 100% and 70%
具体步骤：酶解5-去磷酸化
1. Dilute sufficient CIAP for immediate use in CIAP 1X Reaction Buffer to a final
concentration of 0.01u/μl. Each picomole of DNA ends will require 0.01u CIAP.
(1μg of 1,000bp DNA = 1.52pmol DNA = 3.03pmol of ends.)
2. Purify the DNA to be dephosphorylated by ethanol precipitation, and resuspend
the pellet in 40μl of 10mM Tris-HCl (pH 8.0). Set up the following reaction:
DNA (up to 10 pmol of 5´-ends) 40μl
CIAP 10X Reaction Buffer 5μl
Diluted CIAP (0.01u/μl) up to 5μl
50μl
3. Incubate at 37°C for 30 minutes.
4. Add another aliquot of diluted CIAP (equivalent to the amount used in Step 2), and
continue incubation at 37°C for an additional 30 minutes.
5. Add 300μl of CIAP stop buffer. Phenol:chloroform extract and ethanol precipitate
by adding 0.5 volume 7.5M ammonium acetate (pH 5.5) and 2 volumes of 100%
ethanol to the final aqueous phase.
[b]Note: CIAP may be added directly to digested DNA. Add 5μl CIAP 10XReaction Buffer, 0.01u CIAP/pmol of ends and deionized water to a finalvolume of 50μl (6).
有没有哪位战友配过这两种试剂，能否将经验一起分享一下！因为第一次做相关试验没有经验！谢谢！
eg:1 配好了液体并保存在冰箱里，酶的稳定性如何，能存多久？特别是核酸酶P1
2 酶解组织DNA的反应体系如何确定？具体操作如何？因为查相关文献具体步骤较简单！
