Table 2

The composition of the reaction mixtures, the reagents involved and PCR reaction thermal profiles
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

Gosiewski et al.

Gosiewski et al. BMC Microbiology 2014 14:144   doi:10.1186/1471-2180-14-144

Open Data