the od of the amplified product (if any) is misleading as both primers and dNTPs will absorb at 260 and any stabilising protein and the polymerase will absorb at 280 to give por results.

How much dna template (in ng) are you using in your pcr and what is the source of your dna...plant or animal because if the agarose gel fails then it is probably because the pcr failed and often this is because of using too much dna which has pcr inhibitors mixed in with the dna template

Optimize your PCR conditions by trying different annealing temperatures, MgCl2 concentrations, and template DNA amounts.

Use positive control primers known to work with your DNA template.

Consider using a touchdown PCR protocol to improve specificity.

Check your primer design for potential issues like complementarity or non-specific binding.

Verify your gel electrophoresis setup, including buffer composition and running conditions Anjana Raj

Paul Rutland thank you so much for your suggestion. We have isolated DNA from rodent tails. We have tried two methods (one using kit SciPhi genomic DNA extraction kit and another by referring to a paper). I will check with my PCR conditions again. Primers had been chosen from a paper and I went with the same annealing temperature mentioned in the paper.

Jaishree Sankaranarayanan Thank you for your suggestion. I will check my PCR conditions. I used a new kit Taq Mix (2x) (PCR Master Mix (2x)) from SRL. This primer set has been chosen from a paper. It is a set of sex primers. Gel electrophoresis setup seems fine since the DNA ladder resolved properly.

and how much dna are you amplifying in each reaction Anjana Raj

1. Reagent Issues

• Non-DNAse Free Agarose: Using non-DNAse free agarose can degrade the DNA, resulting in no visible bands. Ensure that the agarose used is DNAse-free1.
• Taq DNA Polymerase: The enzyme may be inactive or degraded. This can happen due to improper storage or handling. Replacing the enzyme with a fresh batch or a different source can help resolve this issue11.

2. PCR Reaction Components

• Missing or Inactive Components: Ensure that all necessary components, including dNTPs, MgCl2, and primers, are added to the reaction mix. Any missing or inactive component can lead to no amplification16.
• Mg2+ Concentration: The concentration of Mg2+ ions is critical for PCR efficiency. Too high or too low concentrations can affect the amplification process. Optimize the Mg2+ concentration for your specific PCR setup10.

3. Primer Issues

• Primer Quality and Concentration: Poor quality or incorrect concentration of primers can lead to failed amplification. Ensure that the primers are of high quality and used at the correct concentration16.
• Primer Design: Poorly designed primers can result in no amplification. Ensure that the primers are specific to the target sequence and do not form dimers or secondary structures4.

4. PCR Conditions

• Annealing Temperature: Incorrect annealing temperature can prevent primer binding and subsequent amplification. Optimize the annealing temperature to ensure proper binding of primers to the target sequence19.
• Cycle Number: Insufficient number of cycles can result in low amplification, making it difficult to detect the product on the gel. Ensure that the number of cycles is sufficient for adequate amplification19.

5. DNA Template

• Quality and Quantity of DNA: Degraded or insufficient DNA template can lead to no amplification. Ensure that the DNA is of high quality and used in the correct amount17.
• Inhibitors in DNA Sample: Contaminants in the DNA sample can inhibit the PCR reaction. Purify the DNA to remove any potential inhibitors17.

6. Gel Electrophoresis Issues

• Gel Concentration: The concentration of the agarose gel can affect the resolution of the DNA bands. Ensure that the gel concentration is appropriate for the size of the DNA fragments you are trying to resolve36.
• Loading Dye and Ethidium Bromide: Ensure that the loading dye and ethidium bromide (or other DNA stain) are added correctly. Missing these components can result in no visible bands16.

Summary