【交流】反向PCR法构建缺失突变

不知道大家做突变的时候都用什么方法。有经验的战友不妨来交流一下啊。说说我的先。由于本人没有这方面的经验，所以**求各位战友的意见和建议啊。大家多多批评指正。
我要做一构建好的质粒上约100bp的片段的缺失，利用反向PCR的方法。以下是利用实验室现有资源的实验设计：
1 Phosphorylation of the 5’-end of a primer

10×kinase buffer 2ul
primer (20uM) 5ul
10mM ATP 2ul
polynucleotide kinase 1ul
dH2O 10ul
Total volume 20ul
37℃ for 30min .Heating to 90℃for 5min .-20℃for later use.

2 Amplification

dH2O 14ul
10×Ex Bufffer 2ul
dNTPs(10mM) 1ul
Kinased Primer1(5uM) 1ul
Kinased Primer2(5uM) 1ul
Plasmid (0.1-1ng/ul) 1ul
Ex Taq 0.25ul
Total Volume 20ul

94℃ 2min
94℃ 45 sec
68℃ 10min

3 End-polishing PCR products with pfu

PCR product 15ul
Pfu (2U/ul) 3ul
dH2O 12ul
30 min at 72℃

4 Purification by Recovery kit

5 Ligation

10×T4 DNA ligase buffer 1ul
T4 DNA ligase 1ul
Purified PCR(4ng/ul) 8ul
Total volume 10ul
20-25℃ for 5hr

6 Transformation