[求救] PCR找不出的盲點
遇到PCR的問題
一開始利用dream tag有P的所要的產物
但之後用相同條件，相同機器
就P不到任何東西
PCR產物約2000bp
PCR配方
水 18ul
10x buffer 2.5ul
10mM dNTP 1ul
primer(F) 1ul
primer(R) 1ul
Dream tag 0.5ul
cDNA 1ul
PCR條件:94度 5min
94 30sec
55/50 30sec
72 2min
72 10min
4
Run 35 cycle
目前試過改進的方法有1.增加cDNA的量變成2ul及3ul
2.降低annealing temperature(EX.55度降為50度)
3.換成2X Master mix的tag
4.換新的cDNA試過~也不行 > <
(sample,primer,水,buffer,PCR machine都一樣)
請教各位厲害人士
還有什麼地方可以改進的??
還是有什麼方法??
謝謝~!
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