1. Lysate Clearance
<ol>
 <li>Grow E. coli cultures transformed with desired plasmid DNA in deep well plates (1 mL of culture/well) in Luria Broth with appropriate antibiotic overnight at 37 &deg;C with shaking.</li>
 <li>Pellet E. coli in culture plate at 5,000 x g for 10 minutes, then decant supernatant.</li>
 <li> Resuspend each pellet in 100 &mu;L Resuspension Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA,100 &mu;g/mL RNase A).</li>
	<li>Add 100 &mu;L/well Lysis Buffer (200 mM NaOH, 1% SDS) and shake using plate shaker for 2 minutes.</li>
 <li> Add 100 &mu;L/well Neutralization Buffer (3.0 M Potassium Acetate, pH 5.5) and shake for 2 minutes.</li>
	<li>Transfer cell lysate to lysate clarification plate.</li>
 <li> Place DNA binding plate on top of 350 &mu;L collection plate and place into bottom of vacuum manifold. Assemble vacuum manifold (see Figure 1).</li>
	<li>Place lysate clarification plate on top of vacuum apparatus.</li>
 <li> Apply vacuum at 10 in. Hg (0.34 bar) and collect filtrate into DNA binding plate. Do not use vacuum greater than 12 in. Hg. (0.41 bar).</li>
	<li>Disassemble vacuum manifold. Place 2 mL waste collection plate in bottom of apparatus.</li>
</ol>
2. DNA Binding
 <ol>
	<li>Move the DNA binding plate to the top of the manifold (see Figure 2).</li>
	<li>Add 300 &mu;L/well Binding Buffer (6 M Guanidine-HCl) and pipette up and down to mix.</li>
 <li>Apply vacuum [~ 5 in. Hg (0.17 bar) for slow vacuum] and discard filtrate. DNA is now bound to membrane.</li>
	<li>Wash with 400 &mu;L/well of Wash Buffer (80% Ethanol).</li>
	<li>Apply vacuum, then discard the filtrate. Discard unnecessary if using 2 mL collection plate or filtering directly to waste.</li>
	<li>Repeat wash once.</li>
	<li>Blot bottom of filter plate on absorbent towel to dry.</li>
	<li>Apply vacuum once more for 5-10 minutes to ensure removal of residual alcohol.</li>
	<li>Blot bottom to ensure removal of ethanol droplets.</li>
 </ol>
3. DNA Elution
<ol>
	<li> Add 70 &mu;L/well Elution Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). If higher DNA concentration is desired, volume can be reduced to 50 &mu;L/well, but the average total yield will be lower.</li>
	<li>Incubate the plate at room temperature for one minute.</li>
	<li>Purified DNA can be eluted by either vacuum or centrifugation.</li>
	<li>Vacuum method: Place clean collection plate (DNase, RNase free) into vacuum manifold. Place filter plate on top of vacuum manifold, apply vacuum at 15 in. Hg (0.5 bar) for 1 minute until all elution buffer has passed through the DNA binding plate. Collect purified DNA (see Figure 3).</li>
	<li>Centrifugation method: Place purification filter plate on top of clean collection plate and centrifuge at 1,000 x g for 5 minutes.</li>
</ol>
4. Quantitative and Qualitative Analyses
<ol>
	<li> Measure OD260/280, calculate concentration of purified DNA.</li>
	<li>Perform agarose gel analysis of the purified pDNA.</li>
</ol>
5. Representative Results
E. coli lysate clarified using traditional centrifugation and Pall AcroPrep Advance lysate clarification Filter Plates showed appreciable reduction of debris from starting material. (Figure 4).
Plasmid DNA at three concentrations was spiked into TE buffer and passed through the AcroPrep Advance lysate clarification filter plate. A comparison of plasmid DNA concentration, pre- and post-filtration, shows ~100% recovery (Figure 5) at all three concentrations.
To demonstrate the recovery of DNA from clarified E. coli lysate, plasmid DNA was spiked into crude lysate and then clarified by filtration. The same plasmid DNA was added to cell lysate following filtration. Electrophoresis was performed on aliquots from each sample (Figure 6). A single, clear band of similar intensity from each sample demonstrates little if any DNA loss during the filtration-based clarification step.
Cultures of E. coli containing pCAT plasmid DNA were purified from E. coli lysates using Pall's 350 &mu;L and 1 mL DNA binding Filter Plates, as well as two other brands of 350 &mu;L DNA binding plates. All purifications were performed as specified in the manufacturer's recommended protocol. The highest DNA concentration and total yield are seen from Pall's 1 mL AcroPrep Advance DNA purification filter plate (Figure 7 and Table 2).
Although Competitor W also shows high DNA concentration (155 ng/&mu;L), total DNA yield is the lowest (4.7 &mu;g/well) due to the low recovery volume, ~30 &mu;L. Competitor M gave the lowest concentration of DNA at 108 ng/&mu;L with a total yield of 5.6 &mu;g/well.
Agarose gel analysis of purified pCAT DNA from the three different DNA binding plates is shown in Figure 8. Each collected sample volume was adjusted to 65 &mu;L to correct for differences in recovery volume prior to electrophoresis. All samples show supercoiled plasmid DNA, but DNA yield from each of the Filter Plates varies.
Figure 9 shows the prepared DNA is all in supercoiled form and has similar concentration.
<img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figure 1" /> Figure 1. Assembly of Vacuum Manifold for Lysate Clarification.
<img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figure 2" /> Figure 2. Assembly of Vacuum Manifold for DNA Binding.
<img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figure 3" /> Figure 3. Assembly of Vacuum Manifold for Vacuum Elution.
<img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figure 4" /> Figure 4. Filtration simplifies sample clarification. Triplicate samples of 300 &mu;L of crude lysate clarified by vacuum filtration (350 &mu;L AcroPrep Advance lysate clarification filter plate) or traditional centrifugation. OD320 measured before and after clarification.
<img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figure 5" /> Figure 5. Full recovery of pDNA after passage through AcroPrep Advance lysate clarification filter plate. 300 &mu;L TE buffer spiked with pCAT at 25, 50 and 100 &mu;g/mL. DNA concentration and recovery calculated from OD260 before and after filtration, N = 2.
<img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figure 6" /> Figure 6. Recovery of pCAT DNA after clarification. Equal amounts of pUC19 spiked into lysate before or after clarification, then diluted 1 to 10 in TE buffer. Loaded 2 &mu;L/lane on 1.2% Agarose gel.
<img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figure 7" /> Figure 7. Higher plasmid DNA yield per well with pall Acroprep Advance DNA binding filter plate than competitor plates. pCAT plasmid DNA yield (OD260) using indicated DNA purification plates with 1 mL (Pall and Competitor M) or 1.5 mL (Competitor W) overnight culture of DH5&alpha;. Purification using plate manufacturer's recommended protocol. Error bars indicate standard error (n &gt; 6).
<img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figure 8" /> Figure 8. Agarose gel analysis of pCAT DNA purified on indicated DNA binding plate. 1.2% agarose gel electrophoresis of the purified pDNA. Pooled samples from three separate purifications, eluate adjusted to 65 &mu;L after purification, diluted 1:10. Loaded 2 &mu;L per lane.
<img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figure 9" /> Figure 9. Quality and recovery of pure dna similar when centrifuge or vacuum methods are used for final eluate collection agarose gel electrophoresis of the purified pDNA. The manufacturer's recommended protocol for AcroPrep Advance Filter Plates was followed for pDNA purification. Elution was performed by centrifuge at 1,000 x g for 5 minutes or vacuum with 15 in. Hg (0.5 bar) for 2 minutes.

The authors of this video-article are employees of Pall Life Sciences who produce the instrument and reagents featured in the video and text.

We describe a facile, streamlined protocol of AcroPrep Advance Filter Plates that affords several advantages. This method provides maximum plasmid DNA yields when processed in manual, semi-automated, or fully-automated AcroPrep Advance formats. An integrated prefilter on the lysate clearance plate provides consistent filtration of samples, even those containing high levels of particulates. The well geometry of the plate results in faster, more uniform filtration rates across the plate with minimal hold-up volume; and the outlet tip geometry provides direct flow of samples into receiver plate without concerns of cross contamination. Importantly, the high binding capacity membrane in DNA binding plate of AcroPrep Advance Filter Plates is optimized for maximum recovery of high quality plasmid DNA.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AcroPrep Advance Filter plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8175 (1 mL) or PN 8075 (350 &mu;l)</td>
<td>3 &mu;m glass fiber 0.2 &mu;m Supor&reg; membrane for clarification</td>
</tr>
<tr>
<td>AcroPrep Advance Filter Plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8132</td>
<td>for DNA Binding</td>
</tr>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 5017</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pressure pump</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 13157</td>
<td>&nbsp;</td>		</tbody>
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streamlined purification of plasmid dna from prokaryotic cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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Video: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...