Team:Tsinghua/Notebook/25 September 2010
From 2010.igem.org
Module Ⅱ, group 4
After getting and then diluting the primer, do Two-step PCR.
PCR with Pyrobest DNA polymerase.
PCR system(50ul):
10×Pyrobest buffer Ⅱ 5ul Pyrobest 0.3ul dNTPmix 1ul Primer1 1μl Primer2 1ul Template DNA 0.5ul Mgcl2 0.5ul ddH2O 40.5ul Total 50μl
PCR program:
Step Condition Time 1 95℃ 5min 2 95℃ 30s 3 58℃ 30s 4 72℃ 1min 5 return to step2 15cycles 6 72℃ 5min 7 add amplification primers 8 95℃ 5min 9 95℃ 30s 10 56℃ 30s 11 72℃ 1min 12 return to step2 30cycles 13 72℃ 1min 14 4℃ HOLD
Run a gel.
result
P+E+M② is positive. It seems that PKC 1-6 are positive. But it still remains unclear whether the PCR bands are so weak and why the digestion result seemed negative.