Plasmid Preparation using Ultrasonication
Ultrasonication is a reliable technique to fragment plasmid DNA. Precisely controllable amplitude, pulsation mode and temperature control are most important features of an ultrasonicator for non-damaging plasmid fragmentation. Additionally, the use of certain agents help to protect against plasmid degradation. Hielscher Ultrasonics offers various solutions for controlled plasmid fragmentation from single vials, simultaneously sonication of numerous samples as well as multi-well plates. Learn more about successful ultrasonic plasmid fragmentation!
Plasmid Shearing using Ultrasonication
When DNA samples are subjected to ultrasonic waves, the ultrasonically generated vibrations create acoustic cavitation in the liquid that shears or breaks high molecular weight DNA molecules via mechanical forces. Sonication is the most widely used method for bulk DNA shearing experiments including applications, such as Chromatin Immunoprecipitation (ChIP), for which small fragment sizes are absolutely crucial to obtain high resolution. (cf. Tseng et al., 2012)
Plasmid DNA (pDNA) is specific form of DNA, characterized by its ring shape and is found in bacteria and some eukaryotes.
Supercoiled pDNA is the desired form of plasmid DNA as it shows best results in down-stream processes such as automated sequencing and transfection. Ultrasonication is suitable to fragment pDNA, including supercoiled pDNA, successfully.
Thompson et al. (2008) demonstrated that plasmid sonication, which is known to fragment supercoiled DNA, is an effective way to improve sequence phred20 read lengths to the point that they are not significantly different from Beckman Coulter’s control template or enzymatically linearized plasmids.
- Precisely controllable
- Reproducable results
- Adjustable to target DNA fragment lengths
- Temperature control
- Scalable to any sample size
Use of Plasmid Vectors
Plasmids are often used as tools to clone, transfer, and manipulate genes. When plasmids are used experimentally for these purposes, they are called vectors. DNA fragments or genes can be inserted into a plasmid vector, creating a so-called recombinant plasmid. Plasmid vectors are used as vehicles to drive recombinant DNA into a host cell and are a key component of molecular cloning.
“Non-viral vectors are being extensively studied for their potential use in gene therapy to treat various complicated diseases. Non-viral vectors protect plasmid DNA against physical, chemical, and enzymatic degradation and deliver the DNA molecule to the target site. For example, cationic liposomes, chitosan and other positively charged nanoparticles form complexes with plasmid DNA through electrostatic interactions. However, the readily formed cationic liposomes/plasmid DNA complexes are relatively large (i.e., 300–400 nm) and heterogeneous in nature making them difficult to use in pharmaceutical applications. The large and heterogeneous plasmid DNA/liposomes, plasmid DNA/aerosols, and plasmid DNA/peptides complexes can be reduced to smaller, and homogeneous particles using ultrasonication.” (Sarker et al., 2019)
A prominent example for the use of plasmid vectors is CRISPR–Cas9. The CRISPR–Cas9 system is typically delivered to cells as a single large plasmid or multiple smaller plasmids that encode a target sequence, a CRISPR guide, and Cas9.
Ultrasonic Preparation of DNA-loaded PLGA Nanoparticles by Nanoprecipitation
Jo et al. (2020) used poly(lactic-co-glycolic acid) (PLGA) in order to form a nanoparticle carrier for the delivery of a model CRISPR–Cas9 plasmid into primary bone marrow derived macrophages. For the nanoprecipitation of PLGA nanoparticles, PLGA with two different end groups (ester and amine groups) were used with the objective that the positively charged amine end caps increase the encapsulation efficiency and loading due to the charge interactions between it and the negatively charged backbone of the DNA. In a 50 mL polypropylene conical centrifuge tube, 100 mg Pluronic F127 was dissolved in 20 mL autoclaved DI water by vortex mixing followed by 30 min of gentle sonication using an ultrasonic bath (see CupHorn). An autoclaved magnetic stirring bar was added and the solution was mixed at 600 RPM for 30 min while the other solutions were made. Plastic labware was used instead of glassware throughout to minimize nonspecific adsorption of DNA. Solutions of PLGA dissolved in DMF (44.48 mg/ ml) and TIPS pentacene dissolved in THF (0.667 mg/ml) were made separately. The PLGA was left quiescently to wet in DMF for 30 min before being sonicated for 30 min. (for full protocol see Jo et al., 2020)
- Extraction of DNA
- Encapsulation of DNA
- Dispersion of nanoparticle-coated DNA
- Delivery of plasmid DNA into cells
Plasmid DNA Protection during Sonication
DNA including plasmids and supercoiled plasmids are highly sensitive degradation. All fragmentation methods available are known for certain disadvantages. Ultrasonic DNA fragmentation is one of the preferred methods since controlled sonication in combination with protective measures allows to reduce shear- and heat- induced damaging of DNA strands.
Besides low amplitude settings, pulsation mode and temperature control during ultrasonic DNA shearing, the use of certain agents showed significant protective effect against DNA degradation. For instance, various polymers, peptides, and lipids protect the plasmid DNA during ultrasonication.
Sarker et al. (2019) demonstrated that when the plasmid DNA / ionic liquid (pDNA/IL) nanostructures were subjected to ultrasonic shear stress for 0, 10, 20, 30, 40, 60, 90, and 120 min and complexed with the commercially available cationic gene delivery agent lipofectamine, the percent of fluorescent positive cells were 80%, 98%, 97%, 85%, 78%, 65%, 65%, and 50%, respectively (see chart below). The percentage of fluorescent positive cells increased when the nanostructures were subjected to ultrasonic shear stress for 10, and 20 min, and thereafter decreased slowly.
Ultrasonic Lysate Preparation
Ultrasonic Cell Lysis Protocol
Start with an enriched sample of cells that was prepared via a cell separation method (e.g., immunomagnetic cell separation, fluorescence-activated cell sorting (FACS), density gradient centrifugation, immunodensity cell isolation).
The cell samples must show a volume of a lysis buffer that is appropriate for the experimental goal and probe-type ultrasonicator.
Hypotonic buffers are preferred as they enhance ultrasonic cell lysis. It is important that additives and salt concentration are used in an appropriate manner.
Select your ultrasonic lysis device: For indirect sonication of vials, the VialTweeter or CupHorn are recommended. For multiwell-plates, the UIP400MTP is the ideal ultrasonicator. And classical probe-type sonication, an ultrasonic homogenizer as the UP100H or UP200Ht with a micro-tip are most suitable.
Protocol for probe-type sonication: Place the ultrasonicator probe into the sample volume in a microcentrifuge tube and sonicate for approx. 10 seconds. Depending on the DNA sample, the sonication might be repeated one or two more times. The required ultrasonic energy input (Ws/mL) depends on the sample viscosity and DNA type. Cooling via ice bath and pulsation mode of the ultrasonicator helps to prevent that the sample is thermally degraded.
After ultrasonic lysis, the sample is centrifuged to separate pellet debris (containing unlysed cells, nuclei, and unlysed organelles)
If the sample is not immediately processed further, it can be can be stored at an appropriate temperature to preserve its viability.
Ultrasonicators for DNA Fragmentation
Hielscher Ultrasonics offers various ultrasound-based platforms for DNA, RNA, and chromatin fragmentation. These different platforms include ultrasonic probes (sonotrodes), indirect sonication solutions for the simultaneous sample preparation of multiple tubes or multi-well plates (e.g., 96-well plates, microtiter plates), sonoreactors, and ultrasonic cuphorns. All platforms for DNA shearing are powered by frequency-tuned, high-performance ultrasonic processors, which are precisely controllable and deliver reproducible results.
Ultrasonic Processors for Any Sample Number and Size
With Hielscher’s multi-sample ultrasonicators VialTweeter (for up to 10 test tubes) and UIP400MTP (for microplates/ multiwell plates) it becomes easily possible to reduce sample processing time due to intense and precisely controllable ultrasonication whilst obtaining the desired DNA fragment size distribution and yield. Ultrasonic DNA fragmentation makes plasmid preparation steps efficient, reliable and scalable. Protocols can be linearly scaled from one to numerous samples by applying constant ultrasound parameters.
Probe ultrasonicators with one to five fingers are ideal for the preparation of smaller sample numbers. Hielscher’s lab ultrasonicators are available with different power levels so that you can choose the ideal ultrasonic disruptor for your DNA-related application.
Precise Process Control
Precisely controllable sonication settings are crucial since exhaustive sonification can destroy DNA, RNA and chromatin, but inadequate ultrasonic shearing results in too long DNA and chromatin fragments. Hielscher’s digital ultrasonicators can be easily set to precise sonication parameter. Specific sonication settings can be also saved as programmed setting for fast repetition of the same procedure.
All sonication are automatically protocoled and stored as CSV file on a built-in SD-card. This allows for accurate documentation of performed trials and makes it possible to revise sonication runs easily.
Via browser remote control, all digital ultrasonicators can be operated and monitored via any standard browser. Installation of additional software is not required, since the LAN connection is a very simple plug-n-play setup.
Highest User-Friendliness during Ultrasonic DNA Preparation
All Hielscher ultrasonicators are designed to deliver high-performance ultrasound, whilst at the same time always being very user-friendly and easy-to-operate. All settings are well-structured in a clear menu, which can be easily accessed via coloured touch-display or browser remote control. The smart software with programmable settings and automatic data recording ensures optimal sonication settings for reliable and reproducible results. The clean and easy-to-use menu interface turn Hielscher ultrasonicators into user-friendly and efficient devices.
The table below gives you an indication of the approximate processing capacity of our lab ultrasonicators for cell lysis and DNA fragmentation:
|Batch Volume||Flow Rate||Recommended Devices|
|vials, small beaker||n/a||Ultrasonic CupHorn|
|up to 10 vials||n/a||VialTweeter|
|1 to 500mL||10 to 200mL/min||UP100H|
|10 to 2000mL||20 to 400mL/min||UP200Ht, UP400St|
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Literature / References
- Mark D. Thompson, Kelly G. Aukema, Dana M. O’Bryan, Stephen D. Rader, Brent W. Murray (2008): Plasmid sonication improves sequencing efficiency and quality in the Beckman Coulter CEQ system. BioTechniques 2008, 45:3, 327-329
- Fykse, Else; Olsen, Jaran; Skogan, Gunnar (2003): Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR. Journal of microbiological methods 55, 2003. 1-10.
- Ming L. Wu; Sindélia S. Freitas; Gabriel A. Monteiro; Duarte M. F. Prazeres; José A. L. Santos (2009). Stabilization of naked and condensed plasmid DNA against degradation induced by ultrasounds and high-shear vortices. Biotechnology Applied Biochemistry 53(4), 2009.
- Sarker, Satya Ranjan; Ball, Andrew S.; Bhargava, Suresh Kumar; Soni., Sarvesh K. (2019): Evaluation of plasmid DNA stability against ultrasonic shear stress and its in vitro delivery efficiency using ionic liquid [Bmim][PF6]. RSC Advances 9, 2019. 29225-29231.
- Miguel Larguinho, Hugo M. Santos, Gonçalo Doria, H. Scholz, Pedro V. Baptista, José L. Capelo (2010): Development of a fast and efficient ultrasonic-based strategy for DNA fragmentation. Talanta, Volume 81, Issue 3, 2010. 881-886.
- Julie Ann Wyber; Julie Andrews; Antony D’Emanuele (1997): The Use of Sonication for the Efficient Delivery of Plasmid DNA into Cells. Pharmaceutical Research 14(6), 1997. 750–756.
Facts Worth Knowing
What are Plasmids?
A plasmid is a small circular DNA molecule that is physically separate from chromosomal DNA and replicates independently. Plasmids are often associated with genes that contribute to the survival of an organism and impart specific advantages, e.g. antibiotic resistance. Plasmids are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. Plasmids are important tools in molecular biology, genetics, biochemistry, and life science. Known as vectors in genetic engineering, plasmids are used to replicate or express certain genes. The targeted alteration of a vector is called vector design.
GFP Analysis in Cell Research
Green Fluorescent Protein (GFP) is a versatile biological marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo. GFP can be excited by the 488 nm laser line and is optimally detected at 510 nm.