Amplification and sequencing of double AB infected individuals

Use the following protocol for specific amplification and sequencing of A and B Wolbachia strains in host individuals carrying a standard A-B double infection.

Since individuals of the same species but coming from different populations can harbor different Wolbachia strains, please always use DNA extracted from a single individual, or from multiple individuals derived from a single female. For each strain perform PCRs using the same DNA for amplification of all five genes. This will assure that all five alleles corresponding to a single strain.

Primers

PCR protocol

This protocol is for guidance only and you may need to optimize it for your particular laboratory.

Perform PCR reactions in a 40 μL final volume using 2 μL DNA, H₂O to volume and reagents at the following concentrations:

Cycling conditions

• 94°C for 2 min
• 37 cycles at:
  • 94°C for 30 s
  • optimal annealing T for 45 s (as specified in the primer table above)
  • 72°C for 1 min 30 s
• 1 cycle at:
  • 72°C for 10 min
  • 4°C hold

After cycling, check that amplification was successful by running 5 μl of each reaction on an agarose gel, with size standards.

Purify the remaining 35 μl and sequence using both forward and reverse primers. A double coverage of each sequence is required.

Use the allele templates provided as reference to verify the exact range of nucleotides for each gene sequence.