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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 2.1 Cloning of IgG protease 2.1.1 Colony PCR 2.1.2 Gel verification 2.2 New IgG protease cloning 2.2.1 Digestion 2.2.2 Ligation 2.2.3 Transformation

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

Cloning of IgG protease

Continued from 9/8 ON plates

Colony PCR

Four clones (A, B, C, D) were picked for colony PCR. Positive control: pSB1C3.BBa_J04450 plasmid. Procedures according to protocol; 1:40 elongation time.

Gel verification

1 % agarose, 90 V, 40 min

Results: Weak and very irregularly sized bands. Probably something wrong with the ligation mixture. New IgGp digestion will be made.

New IgG protease cloning

Digestion

Used pSB1A3.IgGp at a conc. of 139 ng/μl.

10X FD buffer	2 μl
dH2O	3 μl
2 μg DNA	14 μl
FD SpeI	0.5 μl
FD EcoRI	0.5 μl
	20 μl

Incubation: 37 °C, 30 min

Ligation

Used pre-digested (EcoRI & SpeI) pSB1C3 vector from 3/8.

pSB1C3 vector	2.5 μl
IgGp insert	12.5 μl †
5X rapid ligation buffer	4 μl
T4 DNA ligase	1 μl

† Since I used the ligation protocol from 3/8, I mistakenly calculated that 12.5:2.5 would give a 5:1 insert:vector ratio. However, due to the new digestion volume, this actually led to a 12.5:1 ratio.

Transformation

Transformation into 100 μl comptetent Top10.

1. Quick-transformation
   1. 3 μl ligation mix. Incubation 5 min on ice.
   2. Heat-shock 30 sec in 42 °C. Cells cooled on ice.
   3. 100 μl plated onto Cm 25 plate.
2. Standard transformation
   • 3 μl ligation mix.
   • Standard protocol transformation procedures.

Plates incubated ON in 37 °C.