NYMU Taipei/Lab Notes/2007 9 29

From 2007.igem.org

Contents

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





2%Gel check for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS

NYMU Taipei 20070929 TAT OmpRBS check.jpg

  • 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

2%Gel check for IDE&TAT_B

NYMU Taipei 20070929 IDE TATB check.jpg

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