Can you sonicated dna

After fixation, quenching, and washing the crosslinked cell pellet, nuclei can be isolated with a Dounce homogenizer. A Dounce homogenizer or Douncer consists of a glass tube and a pestle. The pestle fits very precisely within the glass tube and moving it can shear the cells, destroying the plasma membrane but leaving the nucleus and organelles intact. However, it is time-consuming and reproducibility from sample to sample or experiment to experiment can be tough and unscalable. Chromatin can also be isolated from cytoplasmic and nuclear preparations using a gradient of sucrose.

In this approach, nuclei are pelleted by low-speed centrifugation while the supernatant contains the cytoplasmic fraction.

Chromatin is insoluble under these conditions and can be recovered by centrifugation. NEXSON consists of two main steps: resuspension of the fixed cell pellet in a buffer compatible with nuclei extraction followed by moderate sonication to isolate nuclei.

In eukaryotic organisms, proteins are synthesized by ribosomes in the cytoplasm and the subset of proteins that are localized to the nucleus are transported there after synthesis.

Without nuclear isolation, both cytoplasmic and nuclear fractions of the protein of interest are present and the cytoplasmic proteins can compete for the antibody binding during the immunoprecipitation step of ChIP assays.

This increases the risk of obtaining a lower signal in the downstream sequencing or qPCR analysis. However, nuclei isolation can be time-consuming e. Including nuclear isolation steps in the ChIP protocol can potentially lead to material loss, and if the starting material is already low there is a risk that there will not be sufficient chromatin remaining after isolating nuclei to perform a ChIP assay.

Therefore, although nuclear isolation will generally improve the quality of ChIP assays, it is not absolutely required and could decrease the quality of data in some cases where the sample amount is limiting.

Fragmentation of chromatin is one of the most crucial steps in ChIP experiments. Different methods exist to shear the chromatin, each with its own set of pros and cons. Sonicators transform electrical energy into ultrasonic energy which is transmitted to the samples being processed.

The sound waves generated by a sonicator create alternating compression and expansion cycles, with rates depending on the frequency of the sound waves. During the low-pressure cycle, high-intensity ultrasonic waves create small vacuum bubbles. When the bubbles can no longer absorb energy, they collapse violently during a high-pressure cycle. This phenomenon is termed cavitation, and it is this cavitation force that results in DNA and chromatin being fragmented during sonication.

A probe sonicator is a simple device that allows sonication of one sample at a time. Probe sonicators are usually quite cheap, making them accessible to almost all laboratories.

An additional consideration when using probe sonicators is that the cooling system is not integrated with the probe, so scientists usually use ice buckets to keep the sample cool during sonication. The inconsistent nature of ice in an ice bucket can create different sample heating profiles between runs and therefore can be a cause of inconsistent results.

Some cooling platforms that maintain the sonicator probe at the exact same depth in each sample every time exist to increase reproducibility sample to sample and maintain a low temperature. Probe sonicators also display a high risk of contamination between samples because between each sample that is sonicated the probe is just rinsed, leaving the potential for some material to be carried over to the next sample being processed.

Furthermore, the tube being sonicated are often open to all allow the probe to access so any hazardous material in the tube could become aerosolized. Finally, and perhaps most importantly, performing sonication for experiments with multiple samples is very time-consuming when using a probe sonicator, and shearing reproducibility is often difficult to obtain.

A multi-sample sonicator allows the sonication of several samples at the same time. Multi-sample sonicators use either water bath sonication technology or focused ultrasonic technology and display an integrated cooling system. This kind of device ensures reproducibility, effectiveness, and temperature monitoring.

However, some multi-sample sonicators are quite expensive, and in particular plates for some commercially-available high-throughput sonicators can cost hundreds of dollars. Enzymatic shearing of chromatin usually uses micrococcal nuclease MNase to digest chromatin into small fragments. MNase displays both exonuclease and endonuclease activity, allowing the digestion of DNA fragment length to as small as one nucleosome.

Enzymatic shearing less aggressive than sonication and thus can protect the epitope of interest so that it can be recognized by the antibody. However, digestion by MNase is biased. Heterochromatic regions of chromatin are also not as accessible to digestion by the enzyme, generating additional bias in the digestion pattern.

Therefore, enzymatic chromatin shearing is not random which many researchers find to be undesirable. When using a single-sample probe sonicator, the probe is directly inserted in the tube and the sonication is direct.

The energy transfer displays high intensity to obtain the desired shearing of chromatin. Processing chromatin using a probe sonicator usually involves diluting the sample in a buffer containing SDS 0.

SDS increases sonication efficiency, chromatin yield, and epitope availability. However, high SDS concentrations can potentially lead to disruption or loss of interactions of proteins that do not directly bind DNA. Since the probe sonicator delivers high energy, the samples need to be kept cold to avoid overheating.

Depending on the material available, you can use either an ice bucket or a more sophisticated cooling platform. The best approach is to use a cooling platform that guarantees constant temperature during the whole sonication process as well as between samples.

When sonicating several samples consecutively, it is important to carefully clean the probe between each sample to avoid cross-contamination. The number of pulses, pulse duration, and pulse intensity must all be optimized for each sample type and ChIP target to achieve the best results.

Keep in mind that if the nuclei are not isolated or the chromatin is very compacted, you will probably need to increase the sonication time or amplitude to achieve efficient sonication.

On the contrary, if your protein of interest is at risk of being degraded, then the sonication time or amplitude should be decreased. Most multi-sample sonicators use either water bath sonication technology e. The main advantages of multi-sample sonicators relative to probe sonicators are the sonication reproducibility, the elimination of cross-contamination, and the faster processing with less hands-on time.

Contrary to probe sonicators, the cooling system for multi-sample sonicators is usually integrated with the device which improves processing consistency between samples. The Bioruptor allows the sonication of up to 16 samples at the same time and the Covaris processes up to 96 samples in each run. However, these two instruments use different approaches to sonicate the samples.

With focused ultrasonic technology used by Covaris sonicators, the sonic waves for each sample are generated by a transducer located beneath the sample, ensuring that the full energy generated by the acoustic bursts is focused on the wells being sonicated. On the contrary, in sonicators that use a water bath, the sonic waves randomly travel in the liquid and the acoustic burst is unfocused, leading to a lack of uniformity between samples.

The frequency of the acoustic waves is low in water bath sonicators, which requires much more energy to efficiently shear cells and therefore generates more heat in the samples. Over-heating is always a concern when performing sonication because it can damage either the DNA or the protein of interest. Focused ultrasonic technology can deliver higher frequency sound waves, reducing the sonication time and heat production.

It is crucial to degas the water before performing sonication with most multi-sample sonicators because any air bubbles or contamination in the water could scatter the acoustic wave and decrease sonication efficiency.

Time of degassing is device-dependent and can take several hours in some cases. Another consideration when using multi-sample sonicators is the cost of consumables. Some sonication instruments are compatible with any kind of tube whereas others are only compatible with their specific proprietary and expensive plates or tubes.

PIXUL uses an array of multiple transducers and lenses to focus ultrasonic energy and can sonicate up to 96 samples at the same time in standard and inexpensive well cell culture plates. Cells can be directly cultured, fixed, and sonicated in a single plate, avoiding the need to transfer material between plates and eliminating the risk of sample loss. The PIXUL sonication platform is therefore the best instrument for high-throughput chromatin shearing.

The PIXUL cooling system is fully integrated into the instrument and the coupling fluid needs only minutes to chill before starting each run no degassing is required. The PIXUL Multi-Sample Sonicator is also ideal for simultaneous sonication of multiple sample types on a single run as well as the optimization of sonication for difficult sample types since 12 different sonication programs can be applied in each run.

This report demonstrated that samples processed with PIXUL were sonicated with greater consistency than the same samples processed with either Covaris or Bioruptor. Contrary to mechanical shearing where nuclear isolation step can be skipped, this step is mandatory for enzymatic fragmentation. As previously mentioned, enzymatic shearing can induce sequence bias in the fragmented chromatin and can be less efficient than sonication because MNase cannot access some regions of heterochromatin. For example, MNase will more easily access open chromatin that is being actively transcribed, so when studying repressive histones modifications such as H3K27me3, mechanical shearing is recommended.

One of the main advantages of enzymatic fragmentation is that the digestion can be fully controlled by changing the incubation time, which can produce specific DNA fragment patterns corresponding to the size of 1, 2, 3 or 4 nucleosomes.

Once the protocol is set up for a cell type, the results are very reproducible. Protein A brings the Tn5 enzyme to specific sites on the genome due to interaction with the antibody. Tn5 cuts the chromatin surrounding the DNA-binding site and inserts Illumina-compatible NGS library adapters so libraries can be generated directly as part of the immunoprecipitation step rather than requiring purification of the ChIP DNA prior to library preparation.

As previously explained, every step in the ChIP workflow, including sonication, must be tested for each cell type and target to achieve the best results.

ChIP assays with standard cell culture samples require between 1, and 5 million cells, depending on the level expression of the target of interest and the nature of its association with chromatin. Although most lysis buffers have buckets of detergent that lyse cell membranes, sonication just gives an extra hand in breaking everything apart. Sonication also breaks up, or shears, DNA in a sample—preventing if from interfering with further sample preparation.

Have you ever noticed when you lyse cells that it can be as thick as syrup? Ultrasound waves transfer energy into your sample, causing turbulence and friction in the liquid. This makes your sample heat up as you sonicate. To prevent your sample heating up too much causing degradation of your precious protein, keep your sample cold. Keep them on ice before and after sonication and preferably during too.

This is about finding a balance between sonicating long enough to make sure everything is sufficiently broken apart but not too much so that you heat up your sample and degrade your precious sample. Many modern sonicators have a pulse mode to reduce heating up of the sample during sonication.

Even with an older sonicator, you can simply turn the sonicator on for 5 seconds, then off for 5 seconds. Repeat 6 times for each 30 seconds. In general, resist the urge to turn it up and blast your sample — lower amplitude for longer will reduce heating of the sample. Has this helped you?