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From 2010.igem.org

Contents 1 Mimmi 1.1 yCCS and SOD 1.1.1 redoing the Site-Directed Mutagenesis 1.2 MITF 1.2.1 Amplifying 2 Andreas

Mimmi

yCCS and SOD

redoing the Site-Directed Mutagenesis

Mix	(µl)	X2 X2		yCCS A		SOD A		conditions
sH2O	40	80		yCCS_mut_F		SOD_mut_F		time	°C
dNTPs	1	2		yCCS_mut_R		SOD_mut_R		30s	95
F primer	1	2		pSB1C3		pSB1C3		30s	95	)
R primer	1	2		~3.5kb		~3.2kb		30s	55	> 22 cycles
DNA	1	2X1						4m	68	)
Pfu buffer	5	10						oo	10
Pfu turbo	1	2
tot	50

SOD Primers
SOD_mut_F 262.55µl H2O --> 100µM
SOD_mut_R 372.64µl H2O --> 100µM
SOD_mut_F 3µl + 24.15µl H2O --> 125ng/µl
SOD_mut_R 3µl + 24.42µl H2O --> 125ng/µl

MITF

Amplifying

Mix phusion	(µl)	/4		Mix Pfu turbo	(µl)	/4			Primers
sH2O	67			sH2O	77				MITF_F
F primer	5			F primer	5				MITF_R
R primer	5			R primer	5
dNTP	2			dNTP	2			conditions
5X buffer	20			10X buffer	10			time	°C
Phusion pol.	1			Pfu pol.	1			2m	98
tot	100	25		tot	100	25		30s	98	)
								30s	40-55	> 5 cycles
								1m30s	72	)
								30s	98	)
								30s	65	> 25 cycles
								1m30s	72	)
								10m	72
								oo	10

Andreas