PCR reactions (25 μL) were performed using 2.5 μL of 10 × buffer (with Mg2+), 2 μL of 2.5 mM dNTP, 0.5 μL of TaqE (2.5U), 2 μL of 5 μM of each primer, 2 μL of undiluted DNA template and 14 μL of ddH2O.
A 0.5 μl aliquot of an undiluted plasmid template DNA was aliquoted into the 48 μl of master mix.
The reaction mixture included 12.5 μL iQ SYBR Green Supermix (Bio-Rad), 1.25 μL of 10 μmol/L solutions of each primer, 7.5 μL autoclaved or filter-sterilized ultrapure water, and 2.5 μL of template (undiluted tick extracts).
For each reaction the following was added to a 96-well plate: 4 µL undiluted DNA ExoSAP-IT cleaned template, 1 µL SNaPSHOT SNP primer mix, 2 µL SNaPSHOT reagent mix, 2 µL sequencing buffer (ABI prisms, Applied Biosystems) and 1 µL MWB.
Preamplification was performed in 15 µl 1×AFLP Amplification Core Mix Module (Applied Biosystems, Foster City, CA, USA) supplied with 0.12 µM Eco+A primer (5'-GACTGCGTACCAATTCA-3'), 0.92 µM Mse+C primer (5'-GATGAGTCCTGAGTAAC-3'), and 2 µl undiluted restriction-ligation product as template.
One μl of undiluted cDNA was used as template for PCR amplification using the GoTAQ system (Promega) under the following conditions: 94°C-two minutes, thirty cycles at 94°C-30 94°C-30, 55°C- one minute, 72°C- one minute, final extenseconds 72°C for ten minutes.
Standard curves for each gene were performed using the undiluted and diluted cDNA to cover the range of all template concentrations.
Undiluted DNA extracted from the flies was used as the DNA template.
DNA from one polyp was prepared with the Chelex method (protocol as described in [ 22]), 1 μl of the undiluted supernatant or 1 μl of a 1 10 dilution was used as template.
To assess the detection limit of the experimental method, DNA templates of 13 streptococcal SAgs were serially diluted 10-fold ranging from undiluted to 10-4.
