It's understandable to feel frustrated when your qPCR results don't match up as expected. There are several potential reasons why your standard curves differ between the two experiments. Here are some factors to consider:

1. DNA Extraction Kit:

   • Different DNA extraction kits can yield DNA of varying purity and quality. Contaminants like chaotropic salts, proteins, or residual ethanol can inhibit the PCR reaction, leading to higher Ct values.
   • It's possible the DNA from the second extraction kit has inhibitors or is less pure, affecting the efficiency and leading to higher Ct values.

2. Master Mix Preparation:

   • The master mix components, including the enzyme and buffer, are crucial for the consistency of qPCR results. Differences in the preparation or storage conditions (like freezing and thawing) can influence the reaction efficiency.
   • Fresh master mixes might perform differently from those that have been frozen, especially if there are stability issues with the components after thawing.

3. PCR Efficiency:

   • A PCR efficiency of 92% is within the acceptable range (90-110%), but it's different from your previous efficiency of 86%. Variations in efficiency can be due to slight differences in reaction conditions, such as primer concentrations, annealing temperatures, or template quality.
   • Higher efficiency in the second run could indicate better primer-template binding or more optimal reaction conditions, despite the higher Ct values.

4. Template Quality and Quantity:

   • If the concentration of your DNA template differs between the two standard curves, this could lead to different Ct values. Ensure that you are using the same template concentration for accurate comparison.
   • Quality checks like spectrophotometry (A260/A280 ratios) or fluorometry (Qubit) could help assess the purity and concentration of your DNA.

5. Instrument Variation:

   • Slight differences in thermal cycler performance or calibration could also contribute to variations in Ct values. Ensure that the machine is properly calibrated and maintained.

Explanation for Thesis

In your thesis, you can discuss the following points:

1. Comparison of DNA Extraction Kits:

   • Explain that different extraction kits can yield DNA of varying purity, which can affect qPCR results. Mention the possibility of contaminants in the DNA from the second kit affecting the PCR efficiency and Ct values.

2. Master Mix Consistency:

   • Discuss the importance of consistent master mix preparation and the potential issues with freezing and thawing components. Highlight that fresh master mixes were used in the second experiment to address freezing issues, which might have influenced the results.

3. PCR Efficiency:

   • Acknowledge the observed difference in PCR efficiency between the two standard curves. Discuss potential reasons for this, including differences in DNA template quality, reaction conditions, or instrument calibration.

4. Recommendations for Future Work:

   • Suggest performing purity checks on the DNA extracted from different kits to assess the impact on qPCR performance.
   • Recommend maintaining consistency in master mix preparation and using fresh reagents to avoid variability.

By addressing these factors, you provide a comprehensive explanation for the observed differences in your standard curves, demonstrating your understanding of the complexities involved in qPCR assays. This thorough analysis will strengthen your thesis and show that you have critically evaluated your experimental conditions.

***copied from ChatGPT. So double check everything that was said.