Table III.
PCR composition. Comparison of the usual PCR reagents quantities per 10μl volume of standard simplex vs. multiplex PCR reaction with or without M13 tailed primers. The fluorescently labeled universal M13 primer was only added when using 5′-M13 universal sequence tailed forward primers for each marker.
| Reagents | μl/simplex | μl/multiplex | Final concentration |
|---|---|---|---|
| 10X PCR Buffer | 1 | 1 | 1X |
| 2.5 mM dNTPs (Fermentas) | 0.8 | 0.8 | 200 μM each |
| 5U/μl Taq (Fermentas) | 0.075–0.1 | 0.075–0.1 | 0.375–0.5 U |
| 5–10 μM primers | 0.24–0.48 | 0.24–0.48 | 0.24 μM each |
| 10 μM Labeled M13 primer | 0–0.24 | 0–0.24 | 0–0.24 μM |
| 25 mM MgCl2 | 0.8 | 0.8 | 2 mM |
| DNA | 50ng | 50ng | 50ng |
| Water | To 10μl | To 10μl | To 10μl |