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

we got the PCR product, about 1kb, just the size we expected.