From 2007.igem.org

Contents 1 PCR for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS 2 PCR for IDE 3 PCR for TAT_B 4 2%Gel check for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS 5 2%Gel check for IDE&TAT_B

PCR for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS

		size	Primer	Template
1	TAT_A	140bp	TAT_F+TAT_A_R	E.coli K12 genome DNA
2	INS_A	111bp	INS_A_F+INS_A_R	MGC clone (H_INS 1)
3	TAT_B	137bp	TAT_F+TAT_B_R	E.coli K12 genome DNA
4	INS_B	135bp	INS_B_F+INS_B_R	MGC clone (H_INS 1)
5	TAT_D	131bp	TAT_F+TAT_D_R	E.coli K12 genome DNA
6	OmpRBS	100bp	OmpRBS_F+OmpRBS_R	pOmpC

Template	1uL
10X buffer	5uL
2.5mM dNTP	2uL
Primer_F	1.5uL
Primer_R	1.5uL
Pfu polymerase	0.5uL
ddH2O	38.5uL
Total	50uL

Step	Temp (°C)	Time (mins)	Cycle
Initial Denaturation	95	10
Denatuation Annealing Extension	95 55 72	1 1 1	35
Final Extension	72	10
Soak	4

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PCR for IDE

	size	Primer	Template
IDE	3105bp	IDE_F+IDE_R	M_IDE

Template	1uL
10X buffer	5uL
2.5mM dNTP	2uL
Primer_F	1.5uL
Primer_R	1.5uL
Pfu polymerase	0.5uL
ddH2O	38.5uL
Total	50uL

Step	Temp (°C)	Time (mins)	Cycle
Initial Denaturation	95	10
Denatuation Annealing Extension	95 55 72	1 1 4	35
Final Extension	72	10
Soak	4

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PCR for TAT_B

		size	Primer	Template
3	TAT_B	137bp	TAT_F+TAT_B_R	E.coli K12 genome DNA

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2%Gel check for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS

• Well 2 TAT_A
• Well 3 INS_A
• Well 4 TAT_B (seems failed)
• Well 5 INS_B
• Well 6 TAT_D
• Well 7 OmpRBS
• Well 8 100bp ladder

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2%Gel check for IDE&TAT_B

• Well 2 100bp ladder
• Well 3 IDE
• Well 4 TAT_B (re-PCR, TAT_B second trail)
• Well 5 1kb ladder