double PCR with tailed primer

Question

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

Answer 1

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