Team:Tsinghua/Notebook/15 July 2010


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Module I, group 2(b)

Ligate E (short for eGFP, the same below) and M (short for mCherry), K (short for Kan) and C (short for Chlr) first at 25℃ for 0.5h.

Ligation system:


 H2O	        6.98μl
 10×buffer	1μl
 eGFP	        0.65μl
 mCherry	0.87μl
 T4 ligase	0.5μl
 Total	        10μl


 H2O	        7.15μl
 10×buffer	1μl
 Kan	        0.57μl
 Chlr	        0.78μl
 T4 ligase	0.5μl
 Total	        10μl

Take 5μl from ligation system to run a gel, other 3μl run a 25μl PCR with each longer fragments’ up-stream and down-stream primers.

PCR system (FastPFU):

 H2O	        13μl
 5×buffer	5μl
 dNTP	        2.5μl
 primer1	0.5μl
 primer2	0.5μl
 template	3μl
 FastPFU	0.5μl
 Total	        25μl


Ligation systems have no result directly running a gel and the PCR products also give a strange outcoming…