Primer test on real tissue
We first tested whether the designed primers (Table 1) could amplify DNA
in simple PCRs conducted on 7 tissue samples of 6 different phyla: a
nematode (Ascaridia galli ), cestode (Raillietina
cesticillus ), insect (Apis mellifera ), arachnid
(Ischyropsalis pyrenaea ), leech (Helobdella stagnalis ),
gastropod (Cepaea hortensis ) and bird (Scolopax
rusticola ). DNA extraction from tissues was performed using the kit
DNeasy® Blood & Tissue Kit (Ref. 69506) (Quiagen). All PCR reactions
(20 µl) were carried out using 1 µl of DNA in reactions with 1.25 µl of
MgCl (25 mM), 2 µl of dNTPs (20 mM), 0.25 of 10X Buffer B, 0.3 µl of
each forward (20 pM) and reverse (20 pM) primers, 1 µl of BSA (10mg/ml)
and 0.25 µl of BIOTAQ™ DNA Polymerase of Bioline (ref. BIO-21060; 5
U/µl). The thermocycle conditions were: 96°C for 1 minute and 35 cycles
of 94°C for 30 seconds, 57°C for 30 seconds, 72°C for 1 minute. PCR
amplification capacity was confirmed by electrophoresis in 1.5% TBE
agarose gel.