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Would I expect salt precipitate on fibres of DNA in a NaCl water solution?

Would I expect salt precipitate on fibres of DNA in a NaCl water solution?


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I just recently conducted an experiment in my biochemistry class where we had to add DNA to distilled water, isotonic saline and 2.5M NaCl. I noticed different physical aspects depending on the solvent, but I'm not sure how I would categorize them. Here's what I saw:

  • Distilled: fibrous precipitate, cloudy liquid
  • Isotonic: Honestly barely noticed a change here? Maybe I wasn't looking hard enough
  • 2.5M: salt precipitate at the bottom and fibres

I know DNA is polar so solubility should increase as the polarity of the solvent increases. But to my recollection what I saw in 2.5M saline vs distilled water did not differ much. However, there is always the possibility that I could have made some experimental errors that led to inaccurate observations.

So, can someone help me deduce the solubility of DNA (simply soluble/not soluble) in each of these solvents, or refer me to a paper I can read? Thanks in advance!


The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up.

It is both interesting and important to understand the reason for some of the steps in the procedure below. An onion is used because it has a low starch content, which allows the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution.


Chromatography

Column chromatography is one of the most common methods of protein purification. Like many of the techniques on this site, it is as much an art form as a science. Proteins vary hugely in their properties, and the different types of column chromatography allow you to exploit those differences. Most of these methods do not require the denaturing of proteins.

To be very general, a protein is passed through a column that is designed to trap or slow up the passing of proteins based on a particular property (such as size, charge, or composition).

There are three main steps to protein purification:

1. Capture. You need to get your protein into a concentrated form. If, for example, you are trying to isolate a protein you have synthesized in an E. coli cell, you could be looking at a protein to junk ratio of 1:1,000,000. For capture purification you need a high capacity method that is also fast. You need a speedy method because your crude solution is very likely to also contain proteases and these can quickly chew up your protein.

2. Intermediate. Intermediate purification requires both speed and good resolution.

3. Polishing. For the final step of purification you need a system that has both good resolution and speed. Capacity is usually irrelevant at this stage.

Some of the more common columns include:

IEX: Ion exchange chromatography. Good for capture, intermediate, and polish.

HIC: Hydrophobic interaction column. Good for intermediate purification.

AC: Affinity chromatography. Good for capture and intermediate purification.

GF: Gel filtration (size exclusion) chromatography. Good polishing step.

Let's look at these types of columns in more detail.

Ion exchange chromatography

Ion exchange chromatography is based on the charge of the protein you are trying to isolate. If your protein has a high positive charge, you'll want to pass it through a column with a negative charge. The negative charge on the column will bind the positively charged protein, and other proteins will pass through the column. You then use a procedure called "salting out" to release your positively charged protein from the negatively charged column. The column that does this is called a cation exchange column and often uses sulfonated residues. Likewise, you can bind a negatively charged protein to a positively charge column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting out will release, or elute, your protein from the column. This technique uses a high salt concentration solution. The salt solution will out-compete the protein in binding to the column. In other words, the column has a higher attraction for the charge of salts than for the charged protein, and it will release the protein in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt, so you will often want to elute with a salt gradient. Different proteins elute at different salt concentrations, so you will want to be sure you know well the properties of your protein best results.

Also be aware that changes in pH alter the charges in proteins. Be sure you know the isoelectric point of your protein (the isoelectric point is the pH at which the charge of a protein is zero) and make sure the pH of your system is adjusted and buffered accordingly.

The basic steps in using an ion exchange column are:

1. Prep the column. Pour your buffer over the column to make sure it has equilibrated to the required pH.

2. Load your protein solution. Some proteins in the solution don't bind and will elute during this loading phase.

3. Salt out. Increase the salt concentration to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. At the end bump the system with a very high salt concentration (2-3M) to make sure all proteins are off the column.

4. Remove salts. Use dialysis to remove the salts from your protein solution.

Temperature doesn't have a huge effect on column chemistry. However, it is better to work cold since proteins are more stable cold.

Hydrophobic interaction chromatography

Where ion exchange chromatography relies on the charges of proteins to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of some proteins. Hydrophobic groups on the protein bind to hydrophobic groups on the column. The more hydrophobic a protein is, the stronger it will bind to the column.

Load the proteins in the presence of a high concentration of ammonium sulfate (not ammonium persulfate). Ammonium sulfate is a chaotropic agent. It increases the chaos (entropy) in water, and thereby increases hydrophobic interactions (the more disordered the water, the stronger the hydrophobic interactions). Ammonium sulfate also stabilizes proteins. So as a result of using an HIC column you can expect your protein to be in its most stable form.

The hydrophobic column is packed with a phenyl agarose matrix. In the presence of high salt concentrations the phenyl groups on this matrix binds hydrophobic portions of proteins. You can control elution of different column-bound proteins by reducing the salt concentration or by adding solvents.

Affinity chromatography.

Affinity chromatography relies on the biological functions of a protein to bind it to a column. The most common type involves a ligand, a specific small biomolecule. This small molecule is immobilized and attached to a column matrix, such as cellulose or polyacrylamide. Your target protein is then passed through the column and bound to it by its ligand, while other proteins elute out. Elution of your target protein is usually done by passing through the column a solution that has in it a high concentration of free ligand. This is a very efficient purification method since it relies on the biological specificity of your target protein, such as the affinity of an enzyme for a substrate.

Gel filtration (size exclusion) chromatography d

Gel filtration, or size exclusion, chromatography separates proteins on the basis of their size. The column is packed with a matrix of fine porous beads.

It works somewhat like a sieve, but in reverse. The beads have in them very small holes. As the protein solution is poured on the column, small molecules enter the pores in the beads. Larger molecules are excluded from the holes, and pass quickly between the beads.

These larger molecules are eluted first. The smaller molecules have a longer path to travel, as they get stuck over and over again in the maze of pores running from bead to bead. These smaller molecules, therefore, take longer to make their way through the column and are eluted last.


Background

DNA extraction kits from silica-based solid phase columns often utilizes a chaotropic buffer that serves both as a protein denaturant and cofactor that promotes NA adsorption. A chaotrope is an ion that disrupts hydrogen bonding and disorders water molecules in an aqueous environment [14]. These ions are ranked within the Hofmeister series by their ability to enhance solubility of proteins. Thus, both hydration interactions and specific ion effects play a key role in dictating the order of the Hofmeister series [15,16]. In simple electrolyte solutions, when controlling for charge, this order manifests itself by the localization of the charge on a given ion [17]. Chaotropic ions exhibit increased charge delocalization that disrupts neighboring hydrogen bonding and directly leads to higher protein solubility in water. Consequently, chaotropic ions in water are usually associated with increased NA denaturation through base-pairing disruptions [18]. Previous work with DNA has shown that adding silica particles to a chaotropic solution will induce a pH-dependent DNA adsorption onto the particle surface [19].

The pH dependence of the DNA-silica-chaotrope adsorption results from the surface charge of silica and DNA [20]. Depending on the column fabrication method, the silica surface may contain a mixture of single silanols and/or geminal silanols with an isoelectric point around pH 1.5–3.6 [21]. Studies have shown the two effective acid dissociation constants (pKa) of silica silanols are pH 4.5 and 8.5 [22,23]. In the context of NA extraction from biological samples at a physiological pH of 7.0–7.4, the vast majority of the exposed silica surfaces should theoretically be covered with negative charges. Moreover, since DNA has an isoelectric point near pH 5, it too is predominantly negatively-charged at physiological pH. Hence, it is very difficult to explain the apparent silica–DNA affinity within NA extraction columns using electrostatics arguments alone [15,16,24]. In terms of microfluidic medical diagnostics research, one must attempt to understand the fundamental surface chemistry by quantifying the interplay among DNA, silica, and chaotropic molecules as a function of pH to fully optimize the NA extraction yield from complicated heterogeneous biological samples [17].

Previous work has examined the DNA-silica-chaotrope interactions at concentrations of DNA in excess of 1 μg/mL [19,25,26]. In these experiments, the high input DNA concentrations often saturated the silica surface [19,26]. Thus, it is very difficult to extrapolate from these results to NA extraction protocols for common clinical samples that do not saturate the silica surface: for example, urine, plasma, and cerebrospinal fluid have DNA concentrations of 40–200 ng/mL, 17 ng/mL, and 3 ng/mL DNA respectively [13,27,28]. Thus, it is important to understand the DNA-silica-chaotrope interaction when the DNA in solution is the limiting reagent. While using miniaturized silica columns with small pores could lead to more efficient adsorption and elution for dilute NA samples, these columns can be susceptible to clogging or surface passivation by other biomolecules such as proteins, lipids and carbohydrates [12,29].

Here, we aim to characterize the DNA-silica-chaotrope interactions, at clinically-relevant DNA concentrations, as a function of pH. The pH values of 3, 5.2, and 8 were chosen on the basis of the pKa values of the silica surface groups, the depurination of DNA (below pH 2), and the dissolution of silica (above pH 8) [30]. DNA adsorption and elution from the silica surface was quantified under conditions that mimic those commonly used in commercial kits and integrated POC diagnostic devices that isolate NAs from human samples [31–33]. However, we incubated the DNA and silica particles significantly longer than most test protocols call for to make sure that equilibrium was reached. To further focus the scope of our investigation, we only considered commercial silica particles made for biological assays, and λ-phage DNA as the input NA. Finally, we restricted the chaotropes used to the guanidinium (CH6N3 or Gu) and thiocyanate (SCN) ions, commonly used in commercial NA extraction kits [4,34].


The Effect of NaCl/pH on Colloidal Nanogold Produced by Pulsed Spark Discharge

A green method, using pulsed spark discharge (PSD) to synthesize colloidal gold, is studied in this thesis. PSD uses spark discharge to synthesize gold nanoparticles (AuNPs) in deionized water (DIW) and/or ethanol (EtOH). While gold nanoparticles have widespread applications in many fields, especially for the human body, in use them must overcome the influence of NaCl and pH value therefore, this study adds NaCl into PSD-AuNPs to simulate the human body to study its stability. Furthermore, a variety of protectants are added in an attempt to determine the best protectant for AuNPs and improve biologically compatible potency. From the results of this study, adding the long-chain-polymer Carboxymethyl cellulose (CMC) or Polyvinyl pyrrolidone (PVP-k30) can prevent nanogold from aggregation and precipitation in NaCl or different pH value and maintain the characteristic of nanogold dispersion by raising the repulsive force between the particles. The results of this study can be a reference of nanogold applying in biomedical science.

1. Introduction

The pulse spark discharge (PSD) method is developed and used to fabricate the AuNPs solution [1–5], which involves a pulse current being passed through two gold electrodes [6–8], which are submerged in deionized water or ethanol. Many methods of producing AuNPs include the introduction of surfactants in order to improve the suspension of the gold particles. However, gold nanoparticles fabricated by the pulse spark discharge (PSD) method in deionized water or ethanol without any surfactants or stabilizers are characterized as a stable colloid, which can be stored for a long term in a glass container at room temperature without visible sedimentation (no apparent precipitate). The DIW_nanogold is safe for the human body, such as target therapy and drug carriers this study will propose the experiments and simulate results of the colloidal gold [9–13] within the NaCl and pH test.

Gold nanoparticles are widely applied in the human body but require overcoming the impacts of NaCl and pH value. Gold number [14] is defined as the amount (mg) of polymer required to prevent the aggregation of 10 cm 3 of gold solution with 1 cm 3 10% NaCl added. This study also proposes an effective gold number method (a more efficient version of Zsigmondy’s [15] method is conducted in the experiment performed here) in order to determine the gold number of potential colloidal gold stabilizers. Instead of varying the amount of surfactant added, increasing amounts of NaCl are added into a solution of colloidal gold with 0.1 mg of stabilizer, thus simulating continuous titrations. Also, the photothermal effect of AuNPs can also be used as a cancer treatment [16]. The superior biological piezoelectric biosensors can be produced, through biocompatibility, the electrical conductivity and the high surface area of nanogold particles [17]. The properties of nanoparticles can apply to metal to produce beneficial reaction of catalytic [18]. This study simulates the human body or normal saline in NaCl in order to discuss the impacts on gold nanoparticles of DIW_nanogold, under various biologically compatible protective agents, for improving biologically compatible potency. The impacts on, and variations in, suspension of pH value of gold nanoparticles (DIW_nanogold, chem._nanogold, and ethanol nanogold) fabricated by other methods, as well as the changes of absorbance and wavelength, are compared.

2. Experimental Setup

This study utilizes the developed PSD system as a preparation method the principle is to use a bar material (Au), which will be generated into nanometal material as top and bottom electrodes there exists no direct contact between the two electrodes thus, there is no physical force produced between the two however, by using electricity converted into heat energy, a kind of hot melting method of electrode rapidly melting is created. The chamber is the main processing center. Deionized water, which has good insulativity, or ethanol, is used as dielectric liquid. The top and bottom electrodes are submerged into dielectric liquid to cause the generated nanoparticles to spread evenly and be directly stored in the dielectric liquid.

2.1. Preparation for NaCl Test

The fourteen agents listed in Table 1 were tested for their ability to maintain gold colloidal suspension. With the simulation of NaCl in proportions similar to those in human body fluids (0.9%), or the proportion of NaCl in normal saline, colloidal gold is mixed with a highly concentrated NaCl solution, which results in

ions attacking the surface electric potential of the AuNPs. With the loss of their zeta potential, the nanoparticles lose their mutual repulsion and agglomerate. In order to avoid the destruction of zeta potential, the use of an adequate protecting agent is required so that the gold colloid can survive in such an ion-rich solution. This study analyzed the impacts of various protecting agents (must be biocompatible for use in medicinal applications) on the agglomerate of PSDAuNPs-DIW and the agent can be used as protecting agent for PSD-AuNPsDIW.

First, 10 mL of the 30 ppm colloidal gold solution is placed into each of the 15 containers, numbered Au(0)

Au(14) then, a second set is prepared by placing 10 mg of the 14 stabilizers into 10 mL of DIW for a 0.1% (w/v) concentration, which are labeled S(1)

S(14). Then, 0.1 mL is taken from each of S(1)

Au(14) so that 0.1 mg of the agent is present. No agents are added into Au(0), the control solution. A mixture of 1 g anhydrous salt, with 10 mL of water, is used for titrations in the amount of 0.1 mL (per round), and color change is noted after each titration. Add 0.1 mL of the 10% NaCl solution in each round. After the tenth round, the proportion of NaCl (1/11) is the amount seen, on average, in human body fluids (0.9%) therefore, the experiment can be stopped at that point. The continuous titrations will show, through level of color change, which stabilizer is the most effective. Figure 1 presents the flow chart of (a) Zsigmondy’s method and (b) presents the more efficient version of Zsigmondy’s method.

2.2. Preparation of pH Test

For ethanol nanogold, ethanol nanogold (ethanol(1/2) + DIW(1/2)), DIW_nanogold, and chem._ nanogold, six bottles of 10ml of each kind are used as a sample group. Each sample group adds HCl for acid tests and NaOH for base tests, and then the change of pH, visual observation of sample group, color changes, and agglomerate are observed. UV-Vis analysis is conducted for nonagglomerated samples and the pH effects on absorbance and wavenumber are analyzed, in order to gain further understanding of the relationships between the suspension of various gold nanoparticles and pH. Figure 2 presents the flow chart of pH effect analysis for DIW_nanogold, chem._nanogold, and ethanol nanogold.

3. Results and Discussion

3.1. Results and Discussion of NaCl Test

Table 2 displays the number of rounds each surfactant required before a major color change was observed. Once one was observed, no more rounds of NaCl were added.

Reference is round 0: if color change is observed after the stabilizer is mixed with colloidal gold, but before any NaCl is added, round 0 is recorded. The statues are analyzed as follows: (1) Nanogold particles which change color just after the salt solution is added: Au(6) and Au(10). (2) Nanogold particles that aggregated, precipitated, or turned white: Au(2) and Au(11). (3) Nanogold particles that turned blue-violet: Au(1), Au(3), Au(4), Au(5), Au(8), Au(9), Au(13), and Au(14). (4) Nanogold particles which did not agglomerate: Au(7) and Au(12).

It is therefore determined that CMC and PVP-k30 solutions provide the best protection against agglomeration in colloidal nanogold. Of these, CMC is the safer choice, as it presents no harm to the human body and is even used in some food items to maintain a food-particle suspension.

3.2. Results and Discussion of pH Test

Chem._nanogold at HCl and NaOH condition, the relation of absorbance and wavelength with pH, as shown in Figures 3(a), 3(b), and 3(c). AuNPs-DIW at HCl and NaOH condition, the relation of absorbance and wavelength with pH, as shown in Figures 4(a), 4(b), and 4(c). Ethanol nanogold at HCl and NaOH condition, the relation of absorbance and wavelength with pH, as shown in Figures 5(a), 5(b), and 5(c). Table 3 shows absorbance and wavelength deviation at different nanogold versus pH.


The Basics of DNA Extraction

You’ve probably heard of the Genetic Code or the Blueprint of Life these terms refer to DNA. All living things, including animals, plants, and bacteria, have DNA in their cells. DNA is a very long molecule made up of a chain of nucleotides and the order of these nucleotides is what makes organisms similar to others of their species and yet different as individuals. Genes are sections within this long DNA molecule.

In order to study DNA, you first have to get it out of the cell. In eukaryotic cells, such as human and plant cells, DNA is organized as chromosomes in an organelle called the nucleus. Bacterial cells have no nucleus. Their DNA is organized in rings or circular plasmids, which are in the cytoplasm. The DNA extraction process frees DNA from the cell and then separates it from cellular fluid and proteins so you are left with pure DNA.

The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.

Step 1: Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. First, mechanical disruption breaks open the cells. This can be done with a tissue homogenizer (like a small blender), with a mortar and pestle, or by cutting the tissue into small pieces. Mechanical disruption is particularly important when using plant cells because they have a tough cell wall. Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins.

Step 2: Precipitation
When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed with mashed up cell parts. Precipitation separates DNA from this cellular debris. First, Na+ ions (sodium) neutralize the negative charges on the DNA molecules, which makes them more stable and less water soluble. Next, alcohol (such as ethanol or isopropanol) is added and causes the DNA to precipitate out of the aqueous solution because it is not soluble in alcohol.

Step 3: Purification
Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol to remove any remaining unwanted material and cellular debris. At this point the purified DNA is usually re-dissolved in water for easy handling and storage.

Targeted Alaska Grade Level Expectations
[9] SC1.1 The student demonstrates an understanding of how science explains changes in life forms over time, including genetics, heredity, the process of natural selection, and biological evolution by recognizing that all organisms have chromosomes made of DNA and that DNA determines traits.


Solvent effects on the catalytic activities of DNAzymes and ribozymes

The use of organic solvents can expand the function of DNAzymes and ribozymes by increasing the solubility of hydrophobic substrates. A relatively low amount of cosolvents (e.g., DMSO at less than 10 vol%) have been used during in vitro selection of catalytic DNA and RNA and in Diels-Alder reactions (Seelig and Jäschke 1999 Chandra and Silverman 2008) and aldol reactions (Fusz et al. 2005) catalyzed by these molecules. The RNA ligation reaction catalyzed by DNA aptazymes in the presence of herbicides (alachlor and atrazine) proceeds more rapidly and in higher yields upon the addition of 10 % methanol, ethanol, DMSO, acetone, or DMF. The cosolvents might facilitate product release, stabilize certain structures, prevent the formation of inactive conformations, and/or improve the association between the DNA and substrates (Behera et al. 2013). Because the catalytically active structures of the DNAzymes and ribozymes are maintained by weak interactions compared with base-paired secondary structures, solvent conditions that significantly disrupt RNA interactions are not suitable for use.

Formation of the structures of natural ribozymes that catalyze site-specific phosphodiester bond cleavage of RNA is accompanied by water release and metal ion binding (Rangan and Woodson 2003 Bevilacqua et al. 2004 Doudna and Lorsch 2005 Lilley 2005 Toor et al. 2008). The effects of osmotic pressure effect on the catalytic activities of a lead-dependent ribozyme and a hairpin ribozyme have been reported. The lead-dependent ribozyme, called the leadzyme, is comprised of a short stem-loop structure (Fig.  5a ) and cleaves RNA in the presence of Pb 2+ . This ribozyme was initially generated by in vitro selection from random sequence libraries (Pan and Uhlenbeck 1992), and the same sequence motifs are found in yeast phenylalanine-specific tRNA and in human mRNAs (Barciszewska et al. 2005). The addition of low-molecular-weight PEG (2.5𠄷.5 vol%) enhances the catalytic activity of an 11-mer leadzyme in the presence of 25 mM Pb 2+ by about a factor of 2 (Giel-Pietraszuk and Barciszewski 2012). This enhancement is attributed to a reduction in water activity that facilitates the release of water during catalysis. Hairpin ribozymes are found in the satellite RNA of plant viruses such as tobacco ringspot virus (Buzayan et al. 1986 Hampel and Tritz 1989). These ribozymes are comprised of a substrate-binding domain loop and a catalytic loop domain (Fig.  5a ), and cleave RNA in the presence of divalent metal ions. As the amount of low-molecular-weight PEG is increased to 10 %, the cleavage rate of an 85-mer hairpin ribozyme in the presence of 1 mM Mg 2+ increases by several fold PEG likely facilitates tertiary folding, which is accompanied by the release of water molecules, while there is no evidence for the dielectric constant effect in this system (Herve et al. 2006).

a The ribozyme structures described in the text and their cleavage sites (red arrows). b Plots of the NaCl concentration dependence of the cleavage rate constant of a hammerhead ribozyme relative to the dependence in the absence of cosolvents (S k c /S k w ) against the dielectric constant (ε r or ε r 𢄡 ) of mixed solutions in the amount of 10 or 20 wt% cosolvents (Nakano et al. 2014b). The curve fits are drawn based on the calculation performed in Fig.  4f . c The reaction cycle for the formation of the catalytically active form of ribozymes in water solution and a mixed solution with a low dielectric constant cosolvent

The hammerhead ribozymes are found in RNA satellites of plant viruses and in genomes including those of humans (Bourdeau et al. 1999 Martick et al. 2008 de la Pena and Garcia-Robles 2010 Hammann et al. 2012). These ribozymes are comprised of three stems (Fig.  5a ), and cleave RNA in the presence of divalent metal ions (Forster and Symons 1987 Uhlenbeck 1987 Haseloff and Gerlach 1988 Martick and Scott 2006 Lee et al. 2008). Cosolvents such as acetonitrile, methanol, DMF, DMSO, ethylene glycol, and glycerol have minimal effects on the rate and yield of the intermolecular cleavage of a 17-mer substrate by a 38-mer ribozyme in 10 mM Mg 2+ but at concentrations higher than 60 vol%, these cosolvents completely inhibit the cleavage reaction (Feig et al. 1998 Mikulecky and Feig 2002). A study using a similar ribozyme motif (intermolecular cleavage of a 15-mer substrate by a 43-mer ribozyme) showed that 20 wt% solutions of ethylene glycol derivatives, small primary alcohols, and aprotic compounds enhance the RNA cleavage rate by several fold in the presence of 10 mM Mg 2+ and by greater than 10 fold at lower Mg 2+ concentrations (Nakano et al. 2009, 2015a). Because the tertiary structure of the ribozyme used in the study is not very stable, the efficiency of Mg 2+ binding is important for the formation of the catalytically active structure and the cleavage rate. The MgCl2 concentration required for rapid catalysis, in which the binding of Mg 2+ is mediated by specific interactions, is changed in the mixed solutions. The concentration of MgCl2 necessary decreases as the dielectric constant of solutions is decreased e.g., the concentration decreases greater than by a factor of 10 in 20 wt% 1,2-dimethoxyethane (ε r =�) than in the absence of cosolvents (Nakano et al. 2015a).

In the reaction catalyzed by the hammerhead ribozyme, divalent metal ions can be replaced by monovalent cations if concentrations are sufficiently high (Murray et al. 1998). Catalysis in the presence of Na + and no divalent cation is mediated by highly cooperative and nonspecific weak interactions with diffusive cations that contribute to folding into the catalytic active form. The NaCl concentration dependence of the cleavage rate constant k (S k = ∂ln k/∂ln [NaCl], in a linear dependence) is changed in the presence of cosolvents (Nakano et al. 2014b). The dependence decreases with the dielectric constant of the solution (Fig.  5b ), and about 40 % less Na + is required in 20 wt% 1,2-dimethoxyethane (ε r =�) than in the absence of cosolvents. Assuming that the substrate cleavage rate is determined by the amount of the catalytic active structure formed, the degree of Na + binding upon formation of the active structure (∆Ψ, which is proportional to S k) is decreased in low dielectric constant solutions (S k c /S k w  <𠂑 and hence ∆Ψ c /∆Ψ w  <𠂑). Thus, the analysis using the reaction cycle in Fig.  5c suggests greater Na + binding to less condensed inactive forms (∆Ψ if) than to the catalytic active form (∆Ψ af), analogous to the case of a duplex formation. About a 40 % decrease in Na + binding in the medium with ε r of about 60 is greater than the decreases observed for duplex formation (60� %) given in Fig.  3 , possibly because of greater Na + -induced collapse upon formation of the active structure of the ribozyme than occurs during duplex formation.

Water-soluble organic polymers are used as macromolecular crowding agents to mimic the crowded environment in cells (Minton 1998). Addition of high-molecular-weight PEG or dextran in the amount of 5� wt% enhances the RNA cleavage activity of group I ribozyme (195-mer RNA), human delta virus (HDV)-like ribozyme (76-mer RNA), and hairpin ribozyme (intermolecular cleavage of a 48-mer substrate by a 35-mer ribozyme) by several fold in a low concentration of Mg 2+ , around 1 mM (Kilburn et al. 2010 Strulson et al. 2013 Desai et al. 2014 Paudel and Rueda 2014). The rate enhancements result from increased compactness of the RNA structures through the excluded volume effect of the polymer additives. In the case of a hammerhead ribozyme motif (43-mer ribozyme and 15-mer substrate), the addition of high-molecular-weight PEG increases the cleavage rate at unsaturated concentrations but not at saturated concentrations of Mg 2+ (Nakano et al. 2009). This study showed that the Mg 2+ concentration required for fast catalysis is decreased in the presence of PEG with an average molecular weight of 8000, as is also the case for smaller cosolvents (Nakano et al. 2015a). Because polymer crowding substantially modifies the water activity and the dielectric constant (Nakano et al. 2004, 2012a), the effects of polymer additives may, at least partly, arise from changes in these solvent properties. Consistent with this possibility, values of ∆Ψ for the hybridization of 15, 25, 33, and 35 base pairs of DNA decrease linearly with the concentration of PEG with an average molecular weight of 3000 (0� wt%) (Markarian and Schlenoff 2010). It has also been reported that PEG with an average molecular weight of 8000 at 20 wt% shortens the length of a single-stranded oligonucleotide but not the length of a duplex (Nakano et al. 2008). In addition, the magnitude of the effect of polymer additives is sensitive to the ionic strength because the cation concentration affects the size of unfolded RNAs (Denesyuk and Thirumalai 2013). These results indicate that polymer solutions have effects on cation binding to nucleic acids in addition to their excluded volume effect.


Minipreps of DNA from Chlamydomonas Cultures

Try Scott Newman’s method, described in Genetics 126:875. I’ve found it quick & reliable. No problems with polysaccharides that I know of.

Here is Scott Newman’s protocol, as currently followed in the Boynton-Gillham laboratory. Cells can be grown as patches on agar plates, or as 1 ml aliquots of cells in multiwell plates.

  • Scrape cells off plate into 1.5 ml Eppendorf tube that contains 0.5 ml TEN buffer.
  • Resuspend vigorously by vortexing, spin for 10 sec. and aspirate off supernatant.
  • Resuspend cells in 150 ul H2O on ice and add 300 ul of SDS-EB buffer, vortex to mix.
  • Extract once with 350 ul phenol/CIA(1:1) for few min by vortexing, separate phases by centrifugation for 5 min., transfer aq. phase to a new tube.
  • Extract once with 300 ul CIA (24:1), transfer aq. to a new tube.
  • Add 2 volumes abs. ethanol, incubate on ice for 30 min., centrifuge for 10 min., wash pellet once with 200 ul 70% ethanol.
  • Dry pellet and resuspend in ca. 40 ul H2O. For Southern analysis use about 1-3 ul.

TEN = 10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl.

SDS-EB = 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl, pH 8.0.

From Mark Buchheim, University of Tulsa
[email protected]

The following is a general mini-prep protocol we have been using to extract both RNA and DNA from green micro-algae (including lots of Chlamys). If you are not interested in the RNA, you may want to opt for a protocol that eliminates the RNA with RNAase.

  • 1. Harvest cells by centrifugation in sterile centrifuge tubes
  • 2. Wash cells in 50mM Tris HCl (pH 9.0). Discard wash.
  • 3. Resuspend and break material in 500 ul lysing buffer (Su and Gibor, 1988, Anal. Biochem. 174:650-657) with ultrasonic probe (10-20 pulses). Check cell breakage by microscopy
  • 4. Add 50 ul Guanidine HCl (8 M) to broken cell extract (precipitates polysaccharide and SDS). Vigorously mix for 1 minute
  • 5. Add equal volume (550 ul) of 49:1 in fume hood
    • mix for 2 minutes
    • spin (at max.) for 2 minutes
    • mix for 2 minutes
    • spin (at max.) for 2 minutes
    • mix for 2 minutes
    • spin (at max.) for 2 minutes
    • 21d. Add 2 volumes (800 ul) ethanol to DNA
    • 22d. Freeze for at least 2 hours or overnight
    • 23d. Pellet DNA by centrifugation (10-20 min, max speed)
    • 24d. Discard supernatant
    • 25d. Dry pellet in DNA SpeedVac caps open
    • 26d. Resuspend pellet in 200 ul water (ART tips)
    • 27d. Sample ready for UV spectrophotometry
    • 28d. Add 2 volumes (ca. 400 ul) ethanol to remaining nucleic acid preps
    • 29d. Freeze for at least 2 hours or overnight
    • 30d. Pellet nucleic acid by centrifugation
    • 31d. Discard supernatant
    • 32d. Dry pellet in DNA SpeedVac caps open
    • 33d. Resuspend nucleic to standard volume (1 ug/ul) in water (ART tips)

    From Tony Palombella, University of Colorado
    [email protected]

    Here’s a protocol I came up with a year or so ago to quickly make up enough genomic DNA for PCR reactions. since then, the Chlamy Newsletter has published a similar miniprep protocol.

    • 1) Resuspend one to several loopfuls of Chlamydomonas in 100 ml of lysis buffer (10mM Tris pH 8.0, 1mM EDTA, 3% SDS) in a microfuge tube, or pellet 1ml of liquid culture 30″ in a microfuge. Remove the supernatant by aspiration and resuspend the pellet in 100 ml lysis buffer.
    • 2) Incubate 15′ at room temperature, mixing occasionally.
    • 3) Add 500 ml TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) in order to help separate phases in subsequent extractions. Add 1/10 volume 3M NaOAc, pH 5.2.
    • 4) Phenol extract once. Chloroform extract twice.
    • 5) Add 1.1 volumes isopropanol. Pellet 15′ in microfuge. Wash with 70% ethanol and air dry. Resuspend in 50 ml TE. A typical yield is 1 – 2 ug of genomic DNA. I use about 5 ul in a PCR reaction.

    More recently, I’ve tried other methods, including:

    • 1) Resuspend a couple of healthy loopfuls of cells in SDS extraction buffer (from Weeks et al. Anal. Biochem. 152:376- 3851986): 2% SDS, 400mM Nacl, 40mM EDTA, 100mM Tris-HCl, pH 8.0. Mix thoroughly, but try not to introduce too many bubbles. The volume used depends on the amount of cells used. Incubate 15′ @ RT or 65 C (depending on how clumpy the cells are).
    • 2) Phenol extract. If the aqueous phase is cloudy, add a couple hundred microliters of water to the tube and spin again.
      Chloroform extract.
    • 3) There’s enough salt in the aqueous phase to isopropanol precipitate directly.

    I get more than a few micrograms of digestable DNA from this protocol, but I haven’t tried it in a PCR reaction.

    I’ve only had problems with polysaccharides when doing CsCl preps. Even then, when I’ve had starch in my preps, a quick spin in a microfuge gets it out of the way after the prep is resuspended.

    From Gernot Gloeckner, University of Freiburg
    [email protected]

    Take 2 ml of a very dense grown culture and spin it down in 2 ml Eppendorff tubes at 3000 rpm.

    Resuspend the pellet in 500 ul of CTAB-Buffer.

      • 2% (w/v) CTAB
        • 100 mM Tris-HCl, pH 8
          • 1,4 M NaCl
            • 20 mM EDTA (pH 8)
            • 2 % (v/v) beta-Mercaptoethanol

            Incubate the solution at 65 degree C for 1 h.

            Extract with 500 ul of Phenol/Chloroform/Isoamylalcohol (25:24:1).

            Take the upper phase and pellet it with 0.7 volumes of isopropanol for 15 min at 4 degree C. Spin down at 12000 rpm for 20 min.Wash the pellet

            From Terri Dunahay, National Renewable Energy Lab

            In response to the request for a miniprep protocol for Chlamy DNA, I have some preliminary information that might help. There was a paper published in Biotechniques recently (Goodwin and Lee, 1993, 15:438-444) “Microwave Miniprep of Total Genomic DNA from Fungi, Plants, Protists, and Animals for PCR”.

            A temporary technician in our lab tried this for DNA isolation from a green alga Monoraphidium. This is a very tough, tiny alga from which it is very difficult to isolate unsheared, clean DNA. In a preliminary experiment using the microwave technique, she was able to isolate about a microgram of digestable DNA from 40 ml of a medium density culture. Unfortunately, she left the lab and we haven’t had a chance to pursue this further, but it looked promising. Granted, its not as “mini” a prep as the one devised by Scott Newman, but it may be more useful for cells that are more difficult to lyse than Chlamy.

            PREPARATION OF DNA USING XANTINE

            For transformant analysis and prep of DNA from wt cells

            1. Centrifuge 5-15 ml of cells for 5 minutes at maximum speed using table-top at 3000 rpm. Use polypropylene 15 ml conical tubes.

            2. Remove medium very well. Cells can be frozen at -70 C at this stage.

            3. Add 2 ml of Xantine buffer and vortex the tube to resuspend cell pellet.

            4. Place the tube in a 65 C water bath and incubate for 40 minutes.

            5. Centrifuge for 5 minutes as in #1. Collect supernatant to fresh 5 ml polypropylene conical tube. Discard pellet.

            6. Add 5 ml of 95% ethanol (2.5 volume ). Mix well. At this point tubes can be stored in -70 C freezer.

            7. Centrifuge 5 min. at 3000 RPM to collect DNA. Discard ethanol. Centrifuge the tubes for 1 minute and remove remaining ethanol with capillary tip. Remove all alcohol.

            8. Resuspend pellet in 300 ul of TE buffer (or water). Transfer the DNA solution to microfuge 1.5 ml tube.

            9. Add 150 ul of 7.5 M ammonium acetate. Mix well by inverting several times.

            10. Add 1000 ul of 95% ethanol and mix well.

            11. Centrifuge for 10 minutes in microcentrifuge. Discard supernatant.

            12. Wash pellet twice with 700 ul of cold 70% ethanol. Centrifuge for 30 sec after last ethanol wash to collect remaining ethanol from the centrifuge tube wall. Remove ethanol with capillary tip.

            13. Resuspend pellet in 300 ul of TE buffer. Add 10 ul of DNase free RNase A and 1 ul of RNase T1. Mix well and incubate for 30 minutes at 37 C.

            14. Add 150 ul of 7.5 M ammonium acetate. Note: Ammonium acetate must be fresh, i.e., not stored more than 1-2 weeks. Mix well by inverting 3-4 times.

            15. Add 1000 ul of 95% ethanol. Mix well by inverting 4-5 times.

            16. Centrifuge for 10 minutes at room temperature. Discard supernatant.

            17. Wash pellet 2 times with 700 ul cold 70% ethanol. Remove last drop of ethanol as described above after last wash.

            18. Resuspend pellet in 20-50 ul of sterilized water. Store in -20C. Concentration of DNA is 2-5 ug/ul. Note: You should expect 750 g DNA from 10 ml of cells. Note: Scale up DNA preparation by using multiple tubes not a larger volumes.

            Potassium ethyl xanthogenate (from Fluka cat # 60040) Synonym: Carbonodithioic, o-ethyl ester. MW 160.3

            RECIPE FOR XANTINE BUFFER:

            Ingredient, Amount/100 ml, Concentration

              • Potassium ethyl xanthogenete, 200.0 mg, 12.5mM
                • 1 M Tris-HCl pH 7.5, 10 ml, 100 mM
                  • 0.5 M EDTA pH 8.0, 2.0 ml, 10 mM
                    • NaCl, 4.09 g, 700mM
                    • Water to 100 ml

                    Adapted and modified from: Methods in Molecular and Cellular Biology 1992, 315-22.


                    CONCLUSIONS

                    Use of the TRIzol supplemented with sarkosyl followed by removal of DNA with the TURBO DNA-free kit (Option 1) is an efficient and effective means of extracting RNA from a diverse array of plants, especially those that are woody, aromatic, or aquatic. With the addition of the traditional CTAB method prior to the TRIzol (Option 2), even the most stubborn taxa were mostly successful and gave consistent RNA quality measures. Option 3, which has been used successfully in many laboratories (including our own, e.g., Buggs et al., 2009 Johnson et al., 2012 ) for RNA isolation, is not the most efficient or robust method for obtaining high-quantity and -quality RNA in transcriptomics across the Embryophyta. Despite the success of the protocols described here, our methods were not successful for some plants that contain high amounts of mucilage, such as Opuntia sp.


                    CONCLUSIONS

                    We have proposed a simple and universal model for the dependence of oligocation ligand concentration needed to induce DNA condensation (EC50) on the DNA (CP) and monovalent salt (Csalt) concentrations. The DNA condensation is well described for a range of conditions, by a model combining the established and uniform salt dependence of the oligocation–DNA dissociation constant with the necessity to neutralize about 90% of the DNA charge. These two assumptions, together with the approximation that the fraction of DNA charge neutralized by bound counterions is dominated by oligocations (NcritNcrit L ), leads to Equation (3). The virtue of this phenomenological relation is that it relates the DNA condensation behavior to its dependence on the experimental conditions in a very clear and simple way. Equation (3) connects two well-studied phenomena of DNA polyelectrolyte solution behavior, namely DNA oligocation-induced condensation and the salt-dependent DNA oligocation binding in a novel way. The appearance of one salt dependent and one salt-independent regime in this condensation follows naturally within this model. This model gives a qualitatively correct prediction of the salt and DNA concentration dependent ε-oligolysine induced DNA condensation. For the simple cations Spd 3+ , Spm 4+ and CoHex 3+ , the description is good in the high salt regime as well as in the low salt regime at higher DNA concentrations (PA data), while the prediction fails in the low-salt and low-DNA concentration limit. To our knowledge, this is the first study reporting a direct and simple connection between oligocation binding to DNA (Kd value) and DNA condensation.

                    Implications to in vivo conditions

                    The dynamic units of the histone–DNA interaction in chromatin are the histone tails with net positive charge from +8 to +14 and average charge density of one lysine or arginine per every three amino acids ( 70). The ε-oligolysines studied in this work can be considered as a simplified model of the histone tails with respect to positive charge and charge density. To a certain extent, the ε-oligolysines with high positive charge, Z = +31 (εK31) and Z = +20 (εRK110, εYRK10, εLYRK10) may serve as a simplified model for the nonspecific interaction between DNA and basic proteins of similar net positive charge (e.g. histones).

                    The concentration of the DNA in the cell nucleus is very high ( 71, 72). Recent fluorescent measurements give average concentration of 116 µM of nucleosomes (CP = 46.4 mM) with the highest concentration 260 µM of nucleosomes in heterochromatin (CP = 104 mM) ( 73). Extrapolating the EZ50 versus Csalt dependencies to such high DNA concentrations ( Figure 7), it is clear that under physiological concentration of salt (K + in the range 50–300 mM), condensation of DNA should always proceed in the salt-independent regime for all natural oligocations (histones, protamines, basic domains of the nuclear proteins, polyamines, etc.). At physiological concentration of salt, the critical degree of DNA neutralization needed to induce DNA condensation would be somewhat lower, in the range Nneut = 0.5 – 0.7, than the higher values Nneut = 0.7 – 0.95 observed at low salt concentration (CKCl = 10–20 mM). In addition, other factors such as the presence of Mg 2+ , polyamines as well as the crowded environment of the cell cytoplasm, would facilitate DNA condensation. Therefore, it is reasonable to suggest that the conditions of DNA in the nucleus are very close to the borderline separating the extended and collapsed DNA phases. Since DNA in the nucleus is in the salt-independent regime of condensation, the principal factor regulating DNA condensation should be any addition/removal of cationic (or any other) components facilitating the DNA collapse. As a result, there exist powerful ‘brakes’ for overproduction of the basic components (polyamines, protamines, histones), such that any excess of these components would result in DNA condensation blocking access to DNA for the transcription/replication machinery. On the other hand, insufficient amount of DNA condensing agents will lead to DNA expansion that might be damaging and harmful in the crowded space of the cell nucleus ( 74). The tight stoichiometry between DNA and the histones has recently been illustrated by the in vivo observation of strong correlation between the amount of the core histones, linker histones and nucleosome repeat length in the chromatin ( 75, 76).

                    Counter-intuitively, the presence of a certain excess of DNA-condensing agents in chromatin may also facilitate access to DNA of the proteins responsible for replication and transcription. From the ITC observation that DNA condensation proceeds with higher affinity than the preceding non-specific (charge neutralizing) interaction, it follows than when the amount of condensing agent exceeds the critical value, the onset of DNA condensation triggers a redistribution of the ligand between the two phases of DNA. Thus, in the region of coexistence between the condensed and extended DNA states, the soluble phase of DNA is depleted of cationic ligands. The remaining uncondensed DNA binds less ligand than under the conditions before the onset of condensation. For the cell nucleus it means that increase in cationic components may lead to DNA condensation (formation of heterochromatin). This will be accompanied by a depletion of oligocations for the rest of chromatin, thus making the euchromatin more open and accessible for transcription. Such an unexpected consequence of excessive concentration of the condensing agents in the chromatin might explain the observation of higher concentration of the polyamines in the eukaryotic cells with high metabolic activity (e.g. in cancer cells) ( 77).

                    Our study demonstrates that the electrostatic mechanism is a universal one and salt-dependence of binding constant (and free energy) of ligand–DNA interaction is expected to be similar for the ligand–DNA interaction in solution and in condensed state. The apparent independence of EC50 on salt concentration simply indicates that the DNA condensation is in salt-independent regime and that dissociation constant of ligand–DNA complex is very low so that KdCP in Equation (3). The net positive charge of the histone octamer forming the basic unit of eukaryotic chromatin, the nucleosome core particle, is about +147 ( 70). Therefore, from the salt dependence of oligocation–DNA-binding constant one can predict that the total electrostatic binding energy of the histone octamer to the nucleosomal DNA (the sum of binging energies of the all basic domains in the octamer) must be significant: ΔGbind el = RTlnKd = RT[lnKd(1M) + ZblnCsalt] ≈ − 150Kcal/mol [assuming Z = +147, Csalt = 0.15M, b = 0.9 and lnKd(1M) ≈ 0]. This estimation demonstrates that the NCP and chromatin in general is an extremely stable polycation–polyanion complex, which challenges the view of a marginal stability of chromatin ( 78). This evaluation for the electrostatic free energy of chromatin formation is a rather low estimate. Some additional favorable contributions to this term might originate from the non-stoichiometric charge ratio between DNA and the histones ( 70, 79, 80). Notably, favorable electrostatic free energy greatly exceeds the unfavorable contributions to the formation of the NCP [mostly due to energy cost of the DNA bending ( 81, 82)] that was confirmed by early experimental observation ( 83) and by theoretical analysis based on the counterion condensation model ( 84). More detailed presentation of the arguments in support of a high stability of chromatin originating from nonspecific histone–DNA interaction as well as the consequence of the high stability of chromatin for its statics and dynamics and for the conditions for the protein machines operating on the chromatin template, is discussed in our earlier work ( 70, 80, 85).


                    Watch the video: How Water Dissolves Salt (February 2023).