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I'm investigating phylogenetic in genus Panax (Araliaceae). I tried some published PCR condition for amplification matK gene with pair of specific primers trnK3914F (5’-TGG GTT GCT AAC TCA ATG G-3’) and trnK2R (5’-AAC TAG TCG GAT GGA GTA G-3’) (attached files) but I could not recognize the desired band. My problem is no observed band. I had tried gradient PCR with Tm ranged from 45 to 68 deegrees also, The size of desired band is 1509 - 1512bp, so I also try to increase the time for strain elongation , but it also was not better.
With another region (ITS1-5.8S-ITS2), it was successful to amplify.
I can not explain why it has failed with matK gene. Please give me advices to modify the PCR condition. Please hint what I should modifify the procedure to do.
Le Ngoc Trieu
I did not use the trnK primer pairs, but matK 390F and matK 1326R. You can also use other pairs like 472F and 1248R or xF and MALPRI. The protocol is the same as the protocol attached using Phusion polymerase. Good luck!