While running on 2% AGE (with product size ~320 bp), the negative control (Plasmid without insert) and the PCR control (without template) show bands with less intensity where there are no bands in the test templates. I have checked the reagents that are used for PCR that we use viz., Premix (GenetBio make), Primers (Sigma), Sterile water (optional to autoclave yet it is done) and they are free from contaminations. The template is fine as well. It is suspected to be pipettes being contaminated yet I wpuld be glad to look from your suggestions. Thanks in advance.
you can use DNAoff for contaminated pipettes and use filter tips for ongoing test to preventing against contamination.
you should change all of your reagents,ideally set up the pcr in another lab with other peoples pipettes and reagents and run the pcr on a thermal cycler not near your working area. It only takes a few molecules of contamination in a reagent , tube or pipette to ruin a reaction.Often this comes from the buffer used in checker gels where some pcr product leaks out of the gel into the buffer and gets transferred on gloves,gel or pipettes into your working area. Ig you cannot get rid of it easily just move one primer outside of the currently amplified area and previous contamination is no longer amplifiable
You can change your tips...... Use filtered tips...
Use commercial reagents.....
Use new buffer..... These light bands are due to old buffer...
and clean your pipettes and working bench with 70% ethanol before starting work. :)
best of luck
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