Hi All,
I am running PCR amplification with fungal primers ITS1 and ITS2. After running the PCR I am getting some samples that have an extra band. My samples are from human dental plaque, and I am not sure if these higher molecular weight bands are possible mammalian ITS region amplification or possibly some sort of bacterial amplification/contamination since my samples are biofilms? I am not seeing the extra band in my positive controls nor am I getting any amplification with my negative control, so I do not believe it is a primer issue. I need to sequence these samples, so if anyone has any insight as to what this band might be it would be appreciated!
Dear Lauren
- 1. Your band appears specific to that sample only suggesting it isnt general PCR conditions
- 2. Accordingly, I would gel extract and sequence
- 3. If you simply wish to work blind and see if this band is non specific and can thus be eliminated by more stringent PCR try raising your annealing temp by 2C and see if you retain the expacted band in all samples but lose the unexpected band in that one sample
Hi,
Extra band might be caused by the following reasons:
1) Primer problems like non-specific bindings, polymerisation, non-optimised PCR conditions etc.
2) Contamination, possible, as your sample was from dental extracts. So you might need to optimize your DNA extraction and purification protocol.
Just try to think back the entire process and try to to trouble shoot every single step.
Good luck!
Regards.
Dear Lauren
- 1. Your band appears specific to that sample only suggesting it isnt general PCR conditions
- 2. Accordingly, I would gel extract and sequence
- 3. If you simply wish to work blind and see if this band is non specific and can thus be eliminated by more stringent PCR try raising your annealing temp by 2C and see if you retain the expacted band in all samples but lose the unexpected band in that one sample
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