From 2007.igem.org
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
|
|
|
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
|
|
|
PCR for TAT_B
|
| size
| Primer
| Template
|
3
| TAT_B
| 137bp
| TAT_F+TAT_B_R
| E.coli K12 genome DNA
|
Check