Anybody have experience with problems with the amplification of Hco/Lco after extraction of fresh specimen from mallaise trap samples? While the amplification of COI (old samples work, so its not a problem concerning the primers - which have been newly ordered) only appears as a smear, other mitochondrial genes like 12S and 16S are amplified without problem. I'd be happy with some advice.
Higher template concentration may results in smear. Optimal template conc is 10 ng for pcr.
Smear also occurs when the samples are diluted 1/5, 1/10 and 1/50... And still, other genes are amplified perfectly without touching the sample concentration.
Hi Jerome, try to find an optimal Mg++ concentration, maybe also the primer annealing temperature is too low. All the best, Nadine
If other genes are showing homology then only they will amplify, even in that case also you can elute the desired band and then go for re-amplification using that as a template. Template concentration and Tm are main cause behind smear. I have tried it....it actually works.
i had that problem too, sometimes i change the Mg concentration and it helps... sometimes we change the primer annealing temperature and it helps too...
First of change your primer set. i had same problem, but after change my primer set, problem was sort out.
When I did my protocol I tryed to change the annealing temprerature , so I used an annealing temprerature of 64,5 °C; then I had to do a purification and extraction of DNA from agarose gels.
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