标准的Touchdown PCR程序
ANNEALING TEMP. 55度
94 5min
94 30s
60 30s
72 1min 2cycles
94 30s
59 30s
72 1min 2cycles
94 30s
58 30s
72 1min 2cycles
........
94 30s
51 30s
72 1min 2cycles
94 30s
50 30s
72 1min 20cycles
72 5min
你也可以做Stepdown
原理同上,就是把降落的梯度增加,以三度为宜。
另一个程序:
94C 4min
94c 5sec
70c 10sec
72c *sec 30个循环,每个循环降0.5c
94c 5sec
55c 10sec
72c *sec 15个循环
72c 7min
延伸时间要看你扩增的片断的长度定,最好先做一个剃度pcr测一下退火温度的范围,然后再做touchdown,这样更有的放矢。
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