Team:Tsinghua/Notebook/17 August 2010
From 2010.igem.org
Module I, group 2(b)
Ligate again from PEM and PC by adding K fragments.
Double digestion system:
kan:
H2O 9μl buffer BamHI 5μl kan 30μl KpnI 4μl BamHI 2μl Total 50μl
PEM, PC:
H2O 30μl buffer BamHI 5μl plasmid 9μl KpnI 4μl BamHI 2μl Total 50μl
Run a gel and cut to purify the digestion products. Measure the concentration. Ligate the PEM and PC with K.
Ligation system:
PEM+K:
H2O 6.5μl 10×buffer 1μl K 1μl PEM 1μl T4 ligase 0.5μl Total 10μl
PC+K:
H2O 4.6μl 10×buffer 1μl K 1.4μl PC 2.5μl T4 ligase 0.5μl Total 10μl
Transform the ligation product into bacterial cells immediately. Spread about 100μl of the resulting solutions on LB plates (with 0.1% ampicillin).
Module I, Group 2c
After purification, ligation reaction took place in a mixed system as following.
eGFP 3ul mCherry 2ul Kan 3ul PSB1C13 2ul T4 ligase(NEB) 1ul T4 ligation Buffer 2ul H2O 7ul
Negative control
PSB1C13 2ul T4 ligase(NEB) 1ul T4 ligation Buffer 2ul H2O 15ul