"

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

P+K+C:

 H2O	         0-10.5μl
 buffer BamHI	 2μl
 P+K+C	         5-16μl
 KpnI	         1.5μl
 BamHI	         1μl
 Total	         20μl

Run a gel. Order: P+E+M 1-6 double digestion, Marker, P+K+C 1-6 double digestion, P+K+C PCR.

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.