different PCR product size with literature

[bestmoment]

Hi,

My PCR product is showing 300bp and 250bp instead of 230bp and 500bp as stated by a literature. All PCR set-up is the same with the reference and it has been repeated twice. My DNA sample is from endophytic fungi of medicinal plant.

Any comment is highly appreciated.

Thanks
nath

[smhall]

• Your negative controls show no bands, right? I am guessing positive controls not possible.
• Keep the prepared PCR reaction tubes on an ice-water bath, and don't transfer to thermal cycler until it is up to initialization temperature (95-100 deg)
• Touchdown PCR to control nonspecific priming
• Template DNA is prepared exactly according to reference, you say?
• Lastly: new set of primers, maybe from another oligosynthesizer (vendor); verify purification method...just desalted...gel/HPLC-purified?

These are just items that would run through my mind.

[Sun of Sevenless]

For the beginning simply cut the products out of gel and sequence with the primers you used for PCR, to make sure those bands are specific. And then you can think about the continue.