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From 2010.igem.org

Module I, group 2(b)

Ligate PEMK with C. Using the PEMK①.

Double digestion system:

chlr:

 H2O	        5μl		
 buffer Tango	2μl		
 chlr	        10μl		
 KpnI	        3μl		
 Total	        20μl	
 37℃ 1h, then	
 + buffer Tango     2.5μl
 + EcoRI	    1μl
   Total	    23.5μl

PEMK:

 H2O	        3μl
 buffer Tango	2μl
 PEMK	        12μl
 KpnI	        3μl
 Total	        20μl
 37℃ 1h, then	
 + buffer Tango     2.5μl
 + EcoRI	    1μl
   Total	    23.5μl

Run a gel and cut to purify the digestion products. Measure the concentration. Ligate the PEM and PC with K.

Ligation system:

PEMK+C:

 H2O	        14.4μl
 10×buffer	2μl
 C	        1μl
 PEMK	        1.6μl
 T4 ligase	1μl
 Total	        20μl

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

Ligation product is transformed in to DH5aα 24h after ligation.