Dear Niaj,

When increasing the volume of a specific component in a PCR reaction, you need to adjust the volume of water to maintain the final reaction volume. For example, if you increase the amount of another component (e.g., DNA), reduce the volume of water by the same amount.

Also, adding 5 µL of DNA template in a total reaction volume of 10 µL may practically not be ideal. I recommend measuring the concentration of DNA in your sample and calculating the appropriate volume to achieve the desired final concentration.

Rajeev Meora Thank you so much for your valuable suggestion

Hello Mr. Neeraj,

A more suitable approach would be to increase the total reaction volume to 20μL. This is actually a more standard reaction volume used in many protocols and offers several advantages. It allows you to increase your template DNA while maintaining proper concentrations of all other components, provides better flexibility in volume adjustments, and ensures adequate water content for optimal reaction conditions. However, before making these adjustments, i would recommend you to check your template DNA concentration, as the issue might be related to DNA quality or quantity rather than volume; second, ensure your primers are working efficiently; and third, verify that your cycling conditions are optimal for your target.

Try it by making 20 μL of PCR mixture, where :

• Master Mix - 10μL/ reaction

• Primer forward - 1μL/ reaction

• Primer reverse - 1μL/ reaction

• Nuclease free water - 7μL/reaction

• DNA template - 1 μL/reaction (may vary)

When increasing the template DNA amount to improve band intensity, you have two main options: adjusting the total reaction volume or the individual component concentrations. Let’s go through both:

Option 1: Keep Total Volume at 10 µL

If you want to keep the reaction volume at 10 µL, increasing the template DNA from 1 µL to 5 µL will reduce the amount of water, so it’s important to ensure the concentrations of other components remain effective. Here’s a suggested approach:

However, since reducing the water volume makes the reaction mixture more concentrated, this approach can sometimes increase non-specific amplification or primer-dimer formation.

Option 2: Increase Total Volume to 20 µL

Increasing the total reaction volume to 20 µL is often preferable, as it allows you to increase the amount of template DNA without changing the concentrations of other components. Here’s how:

This way, you avoid making the reaction too concentrated and maintain optimal conditions for amplification. If you have enough reagents, the 20 µL reaction volume will likely be the most straightforward and effective solution.

Summary

• Option 1 (10 µL): Increase template DNA to 5 µL, reduce water proportionately, and keep other components the same.
• Option 2 (20 µL): Double the total reaction volume and all components to keep concentrations consistent. Zeenat Niaz

The issue with faint bands is *rarely* due to not enough template DNA. In fact, diluting out the DNA to a lower concentration can often solve the faint band problem because you are diluting out PCR inhibitors (proteins, lipids, etc).

Use a 20 micro-liter total reaction volume. You'll need to scale up the dNTPs, buffer, primers etc so they are all at the same final concentration. Or even scale up to 50 microliters.

And, if possible, buy a PCR mix that has all the enzyme, buffer, dNTPS, Mg2+ already combined. Fewer steps = fewer places to make a mistake.

Good luck!

Asif Shahzad

Katie A S Burnette

Abhinav Singh

Rajeev Meora

Thank you so much for your valuable suggestions.

I looked through all suggestions and I would like to note that Katie's suggestion is the most valuable and "professional". This indicates that she has a pretty good experience and so expertise in PCR. Good job, Katie!

Hi Katie A S Burnette ,

may I ask, what is the advantage of doing higher volume PCR? I usually run 10 ul PCR to save material and also to have more uniform temperature during cycling.

Thank you

Larger volumes are more "forgiving" of measuring errors since +/- 1 microliter doesn't matter as much when the volume is increased. The temperature change for volumes that tiny is so quick that 10 vs 20 vs 50 microliters doesn't matter at all. If you want to "scale down", you need to get the reactions working reliably at a standard volume first.

Plus, I've tried it in the past when I was having amplification difficulties. Better to set up a reaction once with a larger volume & have it work than to repeat a smaller reaction a few times. Your time is valuable too!

I would like to add to the Katie's comment. The optimal reaction volume of PCR usually depend on the instrument used. I also like to use small volume PCR. Yes, as you said, it helps to save reagents. Moreover, the smaller the reaction volume - the less problem we have with primer-dimer. For example, so-called "droplet PCR," wherein the reaction volume is measured in nanoliter scale, is free of any mis-amplification, including primer-dimer. From these aspects, I do not see any advantage in using high volume reactions.