18.104.22.168 PCR for species identification
Genomic DNA can be amplified in a 25 µl reaction containing:
- 2.5 μl Taq polymerase buffer (100 mM tris-HCl, pH 8.3; 500 mM KCL)
- 2 U of Taq DNA polymerase
- 1.5-2.5 mM MgCl2
- 250 µM of each deoxyribonucleotide triphosphate
- 0.25 μM of an forward and reverse primer (Table 1)
- 1 µl of DNA
Reaction conditions are as follows:
- initial denaturing for 10 min at 94°C
- 30 cycles of 45 s denaturing at 94°C
- 45 s annealing at 62-65°C
- 1 min extension at 72°C
- final extension for 5 min at 72°C.
Results are visualised using Gel Doc (BioRad) or any other DNA imaging systems. We would recommend that the primer pair 3-F1 and 3-R1 (Table 1) (James and Skinner, 2005) be used as a standard for identification of A. apis. Both primers are A. apis specific as opposed to the primer pair in Murray et al. (2005). Furthermore, 3-F1/3-R1 amplifies a longer PCR product that could be very useful for sequencing efforts (James and Skinner, 2005). The PCR protocol described above can be optimised depending on the type of equipment and reagents used in the laboratory. We would recommend sequencing the PCR product as a quality control, the first time the protocol is implemented in a new laboratory (for more information on sequencing consult the BEEBOOK paper on molecular methods (Evans et al., 2013).