Team:Tsinghua/Notebook/15 July 2010
From 2010.igem.org
Module I, DT and Fan's part:
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:
EM:
H2O 6.98μl 10×buffer 1μl eGFP 0.65μl mCherry 0.87μl T4 ligase 0.5μl Total 10μl
KC:
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
result
Ligation systems have no result directly running a gel and the PCR products also give a strange outcoming…