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I would like to make a double PCR with universal tailed primer. The first PCR would make a tailed amplicon. The second PCR would generate fragment labeled with fluorescent dye. Here is my protcole. Could you tell me if I am wrong ?

  • is my second PCR forward primer well designed to fit with the tail ?
  • What would be the ratio of primer to use if I want to merge the two PCR into one ?

Fist PCR ( works )

In bold, you have the tail M13(-20)

Forward : 5'-GTAAAACGACGGCCAGTTCTGCAGTTGTACTGAGTGAA-3'
Reverse : 5'-AAAGCGTGCAGCTGATATTT-3'

Mix for 1 sample

  • Water = 15.8µL
  • MgCl2 (25mM) = 18µL
  • Buffer (5x) = 6µL
  • dNTP (4mM) = 1.5µL
  • Forward(20pmol/µL) = 1µL
  • Reverse(20pmol/µL) = 1µL
  • GoTaq (5U/µL) = 0.15 µL
  • DNA (50ng/µL) = 1 µL

Second PCR ( not working = no fragment signal )

Forward : 5'-[FAM]-GTAAAACGACGGCCAGT-3'
Reverse : 5'-AAAGCGTGCAGCTGATATTT-3'

Mix for 1 sample

  • Water = 15.8µL
  • MgCl2 (25mM) = 18µL
  • Buffer (5x) = 6µL
  • dNTP (4mM) = 1.5µL
  • Forward(20pmol/µL) = 1µL
  • Reverse(20pmol/µL) = 1µL
  • GoTaq (5U/µL) = 0.15 µL
  • DNA (50ng/µL) = 1µL from my previous amplicon
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  • $\begingroup$ Are you forced to use the reverse like this? I've been taught to avoid A/T ends and stretches of more than 3 consecutive C/Gs or A/Ts; your reverse has both. Besides that, did you try a gradient for the anneaing temperature? $\endgroup$ – Armatus Feb 28 '18 at 9:20
  • $\begingroup$ No, I can change the primer ! Thanks for your advice. $\endgroup$ – dridk Feb 28 '18 at 9:23
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I'm not sure to understand your query. I can say that in my lab we are also using fluorescent dye (for SSR detection). But we are doing it with a one step approach:

Our forward primers are like yours preceded by the fluo sequence (M13-forward primer). But instead of making a second PCR, we add directly the fluo primer (let's called it M13-FAM) in the first PCR. It is REALLY important to put your M13-forward primer in lower quantity than the reverse one (factor 10 for me). To achieve that I use 0.1mM primer solutions and I mix my primers before

For 96 wells (20µL/well)

Primer Mix

  • 1.25 µL of M13-F primer
  • 12.5 µL of R primer
  • 240 µL of water

PCR Mix

  • 200 µL of Primer Mix
  • 20 µL of M13-FAM
  • Master Mix 1mL
  • Water 1mL
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  • $\begingroup$ Thanks ! You answer to my question perfectly ! I was doing 2 PCRs to check my primers first . But my final goal is what you did . Could you give me your primers ? To check if I am not wrong ? I m not sure if M13-tail primer and M13-FAM have to be in same orientation 5'-3' $\endgroup$ – dridk Mar 1 '18 at 16:10

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