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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).