I already tried universal primers (Folmer et al., 1994) and others like COI-E and AnnCOIF used in published papers, with different temperature programs but without results. Does anyone has a PCR protocol that already used and worked sucessfully for these annelids?
Dear author,
I've amplified COI gene from freshwater oligochaetes recently using universal primers too. Electrophoresis showed it is about 600 nucleotides long. Now I'm waiting for sequencing results.
I use the following PCR program: 95°C for 5 min, followed by 35 cycles of 95°C for 40 s, 45°C for 45 s and 72°C for 1 min and a final extension step at 72°C for 8 min. If the material is in good condition it rarely fails. We use a premixed master mix for the PCR reactions and for a 25ul reactrion we use 1 ul of each primer, 2 ul of template, 10 ul mastermix and 11 ul water
I used the following protocol:
1 µl DNA template, 5µl GoTaq Green reaction buffer (Promega), 2.5µl dNTPs (200 µM; Promega), 0.63 µl of both Folmer's primers (0.5 µM), 0.1 µl GoTaq DNA polymerase (0.1 U; Promega) and sterile water up to 25 µl.
Initial denaturation step at 94°C for 4 min followed by 30 cycles of denaturing at 94°C for 45 s, annealing at 45°C for 45 s and extension a72°C for 45 s. A final extension at 72°C for 10 min.
Thank you for your answers... I will try both Svante and Jonathan protocols and see the results :-)
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