【原创】山寨Stratagene“QuikChange Site-Directed Mutagenesis Kits”实验方案
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首先要感谢DXY上的neohjh等几位热心的战友，没有他们的经验我不会这么快做出该实验
引物设计与说明书中不一致，我是参考“An efficient one-step site-directed and site-saturation mutagenesis protocol”这篇文献设计的。
Site-directed Mutagenesis Method
1. Primer design
The primer design was according to “QuikChange® Site-Directed Mutagenesis Kit” and “An efficient one-step site-directed and site-saturation mutagenesis protocol”. The melting temperature (Tm) was calculated with the formula given by the protocol of Stratagene
2. Amplification of mutant PCR
PCR reaction:
DNA template plasmid
0.2 ul
5×PrimeSTAR buffer
10 ul
10 uM oligo 1
1 ul
10 uM oligo 2
1 ul
2.5 mM dNTP
4 ul
ddH2O
33.3 ul
PrimeSTARTM HS DNA polymerase (2.5 U/ul, TAKARA)
0.5 ul
PCR conditions：
95° 60 seconds,18 cycles of 95° 30 s, 55° 15 s, 68° 5 min, followed by incubation at 68° for 10 min.
Product analysis:
The amplification products were check by electrophoresis of 4 μl of the product on a 1% agarose gel.
3. Degradation of parental DNA with Dpn I
The PCRs were purified by Cycle-pure Kit (Omega, China), the PCRs were eluted with 20 μl Nuclease-free water.
Restriction enzyme:
Purified PCR-amplification products
1 ul
10× Buffer TangoTM
2 ul
Nuclease-free water
15.4 ul
Dpn I (10 U/ul, Fermentas)
1.6 ul
Incubate at 37° for 3-4 h.
4. Transformation into E. coli
The digested PCRs were purified by Cycle-pure Kit (Omega, China), the PCRs were eluted with 20 μl Nuclease-free water.
Add 10 ul of digested PCRs into 100 ul DH5α electroporation-competent cells. Plate all aliquot on appropriate antibiotic plate.
5. Detection of Mutants
Miniprep 6 colonies and digest plasmids looking for mutant. Sequence the selected clones to confirm the change.
实验注意事项：
1. 一定要做阴性对照，即不加入Pfu的对照。以便检验Dpn I消化是否完全。转化时还应设置一个阳性对照，即模板质粒。
2. 模板质粒中超螺旋所占比例一定要大于80%。采用质粒试剂盒提取即可。
3. PCR扩增产物即阴性对照一定要经过电泳验证，如果看不到目的条带则应检查PCR程序重新扩增该片段。
4. Dpn I消化体系中扩增产物的量一定要少，一般为1 ul，以保证消化完全，以减少假阳性的出现。
5. 电转化感受态细胞一定要新制，保证高的转化效率。将电转化后的孵育液全部涂在平板上。
6. 观察阴性对照的结果，判定Dpn I消化是否完全。
7. 从每个平板上挑取6个克隆，提取质粒并进行酶切分析。鉴定无误后送交测序。
附件即为所引用的文献
实验中所用试剂：
1. PrimeSTARTM HS DNA polymerase，Takara，购自本地代理商
2. Dpn I ，Fermentas，购自上海生工
3. Cycle-pure Kit，Omega，购自本地代理商
4. 自制电感受态
5. MicroPulser Electroporation Apparatus（电转化仪），美国Bio-rad
6. plasmid mini kit，Omega，购自本地代理商
引物设计与说明书中不一致，我是参考“An efficient one-step site-directed and site-saturation mutagenesis protocol”这篇文献设计的。
Site-directed Mutagenesis Method
1. Primer design
The primer design was according to “QuikChange® Site-Directed Mutagenesis Kit” and “An efficient one-step site-directed and site-saturation mutagenesis protocol”. The melting temperature (Tm) was calculated with the formula given by the protocol of Stratagene
2. Amplification of mutant PCR
PCR reaction:
DNA template plasmid
0.2 ul
5×PrimeSTAR buffer
10 ul
10 uM oligo 1
1 ul
10 uM oligo 2
1 ul
2.5 mM dNTP
4 ul
ddH2O
33.3 ul
PrimeSTARTM HS DNA polymerase (2.5 U/ul, TAKARA)
0.5 ul
PCR conditions：
95° 60 seconds,18 cycles of 95° 30 s, 55° 15 s, 68° 5 min, followed by incubation at 68° for 10 min.
Product analysis:
The amplification products were check by electrophoresis of 4 μl of the product on a 1% agarose gel.
3. Degradation of parental DNA with Dpn I
The PCRs were purified by Cycle-pure Kit (Omega, China), the PCRs were eluted with 20 μl Nuclease-free water.
Restriction enzyme:
Purified PCR-amplification products
1 ul
10× Buffer TangoTM
2 ul
Nuclease-free water
15.4 ul
Dpn I (10 U/ul, Fermentas)
1.6 ul
Incubate at 37° for 3-4 h.
4. Transformation into E. coli
The digested PCRs were purified by Cycle-pure Kit (Omega, China), the PCRs were eluted with 20 μl Nuclease-free water.
Add 10 ul of digested PCRs into 100 ul DH5α electroporation-competent cells. Plate all aliquot on appropriate antibiotic plate.
5. Detection of Mutants
Miniprep 6 colonies and digest plasmids looking for mutant. Sequence the selected clones to confirm the change.
实验注意事项：
1. 一定要做阴性对照，即不加入Pfu的对照。以便检验Dpn I消化是否完全。转化时还应设置一个阳性对照，即模板质粒。
2. 模板质粒中超螺旋所占比例一定要大于80%。采用质粒试剂盒提取即可。
3. PCR扩增产物即阴性对照一定要经过电泳验证，如果看不到目的条带则应检查PCR程序重新扩增该片段。
4. Dpn I消化体系中扩增产物的量一定要少，一般为1 ul，以保证消化完全，以减少假阳性的出现。
5. 电转化感受态细胞一定要新制，保证高的转化效率。将电转化后的孵育液全部涂在平板上。
6. 观察阴性对照的结果，判定Dpn I消化是否完全。
7. 从每个平板上挑取6个克隆，提取质粒并进行酶切分析。鉴定无误后送交测序。
附件即为所引用的文献
实验中所用试剂：
1. PrimeSTARTM HS DNA polymerase，Takara，购自本地代理商
2. Dpn I ，Fermentas，购自上海生工
3. Cycle-pure Kit，Omega，购自本地代理商
4. 自制电感受态
5. MicroPulser Electroporation Apparatus（电转化仪），美国Bio-rad
6. plasmid mini kit，Omega，购自本地代理商
