I carried out PCR with ITS region specific primers on Tsetse fly derived DNA samples and got some bands as shown in the image shown....when i repeated the PCR with tagged primer for sequencing, I got no bands except for the last two lanes of positive controls. What could be the reason?
Hello Alex,
As Sergio has said, your primers are not so good. I had set several NGS libraries by PCR, and the tagging primers did not have side-effects. I guess the shortest band are your primers in the picture, they are or bright, so this means your primers can bind to each other.
Hope helpful to your!
Bonjour Alex
je suis de l'avis de notre ami Sergio, je pense que tes amorces marquées formes quelque part des dimers et du coup elles ne se fixent pas la ou elles devraient. vérifie aussi les températures de fusions de tes amorces ou bien essaye de rajouter du glycérol dans ton mix.
j’écris en français car mon anglais n'est pas fameux.
j'espère que cela pourra t'aider
Thank you Sergio, Falseplanet and Blaid. I realized Tm changes after the tagging....I will do a serial to find out the best annealing temp. to use.
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