From: A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood
NESTED multiplex qPCR | Multiplex qPCR | ||||
---|---|---|---|---|---|
[final volume 50 μl] | |||||
I amplification | II amplification | ||||
[final volume 25 μl] | [final volume 10 μl] | ||||
1. H2O | 6,7 μl | 1. H2O | 2,08 μl | 1. H2O | 0,4 μl |
2. Buffer B | 2,5 μl | 2. Buffer B | 1,0 μl | 2. Buffer B | 5,0 μl |
3. EXT_BAC_F | 0,125 | 3. GN/GP_F | 0,2 μl | 3. GN/GP_F | 1,0 μl |
4. EXT_BAC_R | 0,125 | 4. GN/GP_R | 0,2 μl | 4. GN/GP_R | 1,0 μl |
5. EXT_FUN_F | 0,125 | 5. GP_probe | 0,05 μl | 5. GP_probe | 0,25 μl |
6. EXT_FUN_R | 0,125 | 6. GN_probe | 0,05 μl | 6. GN_probe | 0,25 μl |
7. dNTP’s | 2,5 | 7. FUN_F | 0,2 μl | 7. FUN_F | 1,0 μl |
8. MgCl2 | 2,5 | 8. FUN_R | 0,2 μl | 8. FUN_R | 1,0 μl |
9. Polymerase Perpetual Taq | 0,3 | 9. Asperg_prob | 0,05 μl | 9. Asperg_prob | 0,25 μl |
10. DNA | 10 | 10. Candid_probe | 0,05 μl | 10. Candid_probe | 0,25 μl |
11. dNTP’s | 1,0 μl | 11. dNTP’s | 5,0 μl | ||
12. MgCl2 | 1,8 μl | 12. MgCl2 | 9,0 μl | ||
13. Polymerase Perpetual Taq | 0,12 μl | 13. Polymerase Perpetual Taq | 0,6 μl | ||
14. DNA (product of I amplification) | 3,0 μl | 14. DNA | 25,0 μl |