How To Optimize Your Cell Lysis Method

Start your sample prep with a reliable, reproducible cell lysis method

Cell lysis is the first step to getting the DNA, RNA, proteins, or other analyte you are interested in analyzing from your sample. Because the specific protocol you use can heavily influence the quality, yield, and representativeness of your extracted molecules, it is critically important to devote time to optimizing your method.

There are many different methods of cell disruption to choose from, and the ideal choice depends on a variety of factors, such as sample type and downstream application.

Optimizing your method may require any or all of the following activities:

• Ensuring a clean working environment that mitigates degradation and contamination
• Selecting and comparing different methods of cell disruption 
• Selecting an appropriate lysis buffer 
• Identifying the ideal temperature for cell disruption
• Adjusting sample volume, processing speed, and incubation times, and other variables within your protocol

Protect your molecules and mitigate contamination

When you get to the end of your extraction method and go to assess the quality and concentration of your DNA, RNA, or protein, you want to see a strong, clean gel band; a “just-right” A₂₆₀/A₂₈₀ reading on the spectrophotometer; and/or a beautifully blue hue in your Bradford solution.

Unfortunately, this is not always the case. DNA, RNA, and proteins are vulnerable to degradation, denaturation, and contamination once cells are lysed because they get exposed to endogenous, and potentially exogenous, DNases, RNases, proteases, and various particulates. 

With the right preparation and mitigation techniques, you can maximize your yield and purity so you can smoothly move on to the next phase of your experiment.

ProTips for protecting your molecules prior to and throughout cell disruption:

• Thoroughly clean your bench, equipment, and glassware with an alcohol-based solution, suitable laboratory cleaning products, or RNase Erase (for RNA handling)
• Use DNase- and RNase-free tubes and pipette tips
• Wear gloves and PPE throughout your cell lysis and extraction methods
• When appropriate, add reagents that inhibit RNases, DNases, and proteases to your lysis buffer
• Consistently use fresh pipette tips—avoid using the same tip between samples and from samples to buffers and reagents 

Choosing between the different methods of cell disruption

Certain cell lysis methods are better for specific sample types. For example, osmotic and chemical lysis is suitable for bacteria, yeast, and fungi, whereas bead beating is ideal for tough plants (and virtually any type of sample).

Below are common cell lysis methods and within each of the five categories below, there are several factors to determine. For instance, when using bead beating, you can optimize your method by selecting the appropriate lysing matrix material, size, and shape.

Selecting and adjusting your lysis buffer

Suitable lysis buffers and the specific reagents you’ll need will vary based on your sample type, target analyte (nucleic acids vs. proteins), and downstream application. 

Commercial extraction kits, such as MP Bio’s DNA and RNA isolation kits, will provide the appropriate lysis buffer—optimized for the intended sample types. In the case that you are not using a kit and are homebrewing your buffer, you may want to consider testing different types of lysis buffers on a small subset of samples.

ProTips for labs homebrewing lysis buffers:

• Test the pH and adjust it as necessary—the pH of your buffer is critical to the success of your cell lysis method. If you are using liquid-liquid extraction methods based on density gradients, the pH helps position different analytes in certain phases of the solution gradient. An incorrect pH could result in your target molecules going to an unexpected phase that ends up getting discarded.
• Modify and test standard protocols—while certain lysis buffers are more suitable for mammalian cells versus plant tissues, not all plant tissues are alike and you may need to adjust common methods to generate your ideal protocol. We suggest looking to the literature when you have unique or particularly difficult samples to see if your peers have developed a modified solution that is effective for samples similar to yours.    

Adjust key variables within your protocol

The different methods of cell disruption have variables you can tinker with to optimize your protocol, but here are some common variables to start with:

• Lysis temperature—many methods cause samples to overheat or facilitate rapid RNase, DNase, or protease activity. Keeping the samples on ice or in cryogenic conditions can help slow enzymatic activity and keep your molecules stable
• Incubation times—the amount of time your cells incubate in lysis buffer can influence product yield. In many cases, it may be better to incubate your samples overnight, but keep in mind that you may need cooler temperatures for longer incubation periods
• Speed and cycle settings—cell lysis often involves vortexing or some sort of agitation. Altering the speed of agitation, time between agitation, and total agitation time can influence product yield

Remember, good data starts with good sample preparation, so take the time to optimize your protocol.