Hi everyone,
- I am working on T-ARMS PCR to detect rare BRCA1/ BRCA2 variants in my research. However, I am facing an issue with non-specific band amplification for most of the variants only when I am doing MgCl₂ optimization. Even negative controls are posititve for heterozygous mutant.
Experimental Details:
- Primer Design: No ismatches introduced at 3' terminal for inner primers. Primer specificity was already checked using In silico PCR and NCBI BLAST.
- Annealing Optimization: A gradient PCR was performed with 1.5mM MgCl₂. NO non-specific bands were observed after adjusting primer concentration. 52.4 was selected as the optimum.
- MgCl₂ Optimization: Tested a range from 0.5 mM to 1.5 mM, but non-specific bands remain. I have attached the gel images, with the used concentrations in the labelling.
- Primer Concentration: Currently using an inner:outer primer ratio of 1:2.
- PCR Conditions: Standard conditions with a final reaction volume of 25 µL.
Despite multiple optimization attempts, the non-specific band remains.
I would greatly appreciate any suggestions on how to improve specificity and eliminate unwanted bands.
Thank you in advance!
Bands in your negative controls means you have contamination. It happens, but you should be able to get rid of it by a really deep clean. Focus on getting that eliminated and then you can optimize your assay.
Throw away ALL of your reagents (water, buffer, primers, taq mix, etc.). The only thing you are saving is the DNA. Make everything new - open a new tube of taq mix, make new TE buffer for resuspending primers.
Get all new plasticware & disposables (tips, tubes, tissues, etc.). Wipe your bench area and remove anything that isn't absolutely needed.
The most common cause of contamination is from your micropipettes. Clean them really well, don't use the same micropipette to load gels that you use for setting up PCR.
Good luck!
Thank you Katie A S Burnette. However, I ran a no template control run to eliminate contamination and got no bands, indicating no contamination.
Hi Kavinya Rajan the contamination in negative controls indicates an issue that must be resolved before assay optimization. Perform a thorough decontamination of the workspace, equipment, and surfaces. Discard all reagents, including water, buffers, primers, and taq mix, while retaining only the DNA. Use entirely new reagents, prepare fresh TE buffer for resuspending primers and open a new tube of taq mix to ensure reliability.
For the primer Both inner and outer ration keep it 1:1, it works perfectly for me in ARMS pcr as 1:1 ratio, and whats the ptimer concentration you are using for your 25ul reaction ? make its optimise also for better result. Also chek whether the primer forming primer dimer and its faint and coming down to your target region
@Sandeep Kumar Swain Thank you for your response. I further optimized the [primer] and a inner:outer primer ratio of 1:5 is working best for me currently. I am using a working concentration of 0.5 and 0.1 for outer and inner primers, respectively.
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