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From 2010.igem.org

Module I, DT and Fan's part:

Double digestion of the eGFP, chlr and pUC19 for each pair of restriction sites. Digest at 37℃.

Double digestion system:

eGFP:

 H2O	        2μl
 buffer Tango	4μl
 eGFP	        10μl
 SalI	        2μl
 HindIII	2μl
 Total	        20μl

pUC19 for E:

 H2O	        10μl
 buffer Tango	4μl
 pUC19	        2μl
 SalI	        2μl
 HindIII	2μl
 Total	        20μl

chlr:

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

pUC19 for C:

 H2O	        13μl		
 buffer Tango	2μl		
 pUC19	        2μl		
 KpnI	        3μl		
 Total	        20μl  
 37℃ 1h, then	
 + buffer Tango    2.5μl
 + EcoRI	   1μl
 Total	           23.5μl

Productions purify and measure the concentration. Ligate with NEB T4 ligase at 25℃ for 1h.

Ligation system:

 H2O	        1.2μl
 10×buffer	1μl
 fragment	2.3μl
 plasmid	5μl
 T4 ligase	0.5μl
 Total	        10μl

Transform the ligation product into bacterial cells immediately. Spread about 200μl of the resulting solutions on LB plates (with 0.1% ampicillin).