I am getting a non specific band in PCR product using ITS3 and ITS4 at annealing temperatures of 56,57,58 and 59oC. Can anybody help?

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Nandagopal Murugan

Dear Gulnaz,

This could be due to the DNA extraction from the Isolate or Specimen.

Further reduce the Concentration of Template DNA added.

Check with other Positive controls such as Aspergillus niger, Fusarium etc.,

For further details contact me at [email protected]

René Köppel

Hi Gulnaz Bashir

Try a step down cycling protocoll comming from 65°C lowering the annealing temperature each cycle for e.g. 0.5°C down to 55°C. Antibody may help for concentration of the target fungi. But for this I dont have the experience.

Success

Gulnaz Bashir

Thank you Nandagopal and Koppel for your suggestions.