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