NYMU Taipei/Lab Notes/2007 9 29

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PCR for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS
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<p>
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<h4>PCR for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS</h4>
{| align=left | border=1 | style="text-align:center"
{| align=left | border=1 | style="text-align:center"
!
!
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|1.5uL
|1.5uL
|-
|-
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|pfu polymerase
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|<i>Pfu</i> polymerase
|0.5uL
|0.5uL
|-
|-
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|-
|-
|}
|}
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</p>
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<br>
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<p><hr>
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<h4>PCR for IDE</h4>
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{| align=left | border=1 | style="text-align:center"
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!
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!size
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!Primer
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!Template
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|-
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|IDE
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|3105bp
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|IDE_F+IDE_R
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|M_IDE
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|-
 +
|}
 +
 +
{| align=right | border=1 | style="text-align:center"
 +
 +
|Template
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|1uL
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|-
 +
|10X buffer
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|5uL
 +
|-
 +
|2.5mM dNTP
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|2uL
 +
|-
 +
|Primer_F
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|1.5uL
 +
|-
 +
|Primer_R
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|1.5uL
 +
|-
 +
|<i>Pfu</i> polymerase
 +
|0.5uL
 +
|-
 +
|ddH2O
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|38.5uL
 +
|-
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|Total
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|50uL
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|-}
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 +
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{| align=center | border=1 | style="text-align:center"
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!Step
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!Temp (°C)
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!Time (mins)
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!Cycle
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|-
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|Initial Denaturation
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|95
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|10
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|
 +
|-
 +
 +
|Denatuation<br>Annealing<br>Extension
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|95<br>55<br>72
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|1<br>1<br><font color=red>4</font>
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|35
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|-
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 +
 +
|Final Extension
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|72
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|10
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|
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|-
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|Soak
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|4
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|
 +
|
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|-
 +
|}
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</p>
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<br><br>
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<hr>
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<p>
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<h4>PCR for TAT_B</h4>
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{| align=left | border=1 | style="text-align:center"
 +
!
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!
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!size
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!Primer
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!Template
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|-
 +
 +
|3
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|TAT_B
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|137bp
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|TAT_F+TAT_B_R
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|E.coli K12 genome DNA
 +
|-
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|}
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<br><br><br>
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<hr>
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<h4>2%Gel check for TAT_A INS_A TAT_B INS_B TAT_D OmpRBS </h4>
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[[Image:NYMU_Taipei_20070929_TAT_OmpRBS_check.jpg|300px]]
 +
*Well 2 TAT_A
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*Well 3 INS_A
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*Well 4 TAT_B (seems <font color=red>failed</font>)
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*Well 5 INS_B
 +
*Well 6 TAT_D
 +
*Well 7 OmpRBS
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*Well 8 100bp ladder
 +
 +
<hr>
 +
 +
<h4>2%Gel check for IDE&TAT_B</h4>
 +
[[Image:NYMU Taipei 20070929 IDE TATB check.jpg|300px]]
 +
*Well 2 100bp ladder
 +
*Well 3 IDE
 +
*Well 4 TAT_B (re-PCR, TAT_B second trail)
 +
*Well 5 1kb ladder

Latest revision as of 09:42, 2 October 2007

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

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