This research was supported by the Microbial Ecology Collaborative with funding from NSF award #EPS-1655726.

1. Laboratory bench and tool preparation for loading a 96-well plate
<ol>
	<li>Clear bench top by misting with 70% ethanol. Wipe and let air dry before spraying bench top with 10% bleach. Wipe the bench top dry.</li>
	<li>Sterilize micro-scoopula, spatula, and surgical scissors (curved) by dipping in 95% ethanol and then expose to a flame. Dip each in 10% bleach and allow to air dry prior to use. 
	NOTE: Tools should be sterilized between each sample.</li>
</ol>
2. Sub-sampling and sample preparation
<ol>
	<li>Sterilize gloves using ethanol prior to sub-sampling.</li>
	<li>Homogenize soil samples thoroughly prior to sub-sampling.</li>
	<li>Using sterilized tools, load a labeled 2 mL centrifuge tube with sample until approximately&#160;half full.</li>
	<li>Repeat step 2.2 until all 95 samples have been sub-sampled into labeled 2 mL centrifuge tubes. The 96th well should be used as an extraction blank. 
	NOTE: Steps 2.3 and 2.4 are done to minimize required storage space and to prevent over or double sampling.</li>
	<li>Store 2 mL sub-sample tubes on ice until loading of plate.</li>
	<li>Label 96 sterile 200 &#181;L flat-capped PCR tubes A1&#8210;A12, B1&#8210;B12, &#8230;. H1&#8210;H12. Place these tubes in order into a 96-well rack.</li>
	<li>Assign a sample ID with a well location (A1&#8210;H12) and record it.</li>
</ol>
3. Plate preparation
<ol>
	<li>Remove the rubber cover from 96-well plate (Figure 1A&#8211;1C) and place it into a sterile plastic bag (see Table of Materials). Seal the plastic bag to prevent contamination.</li>
	<li>Cover the 96-well plate with sealing film (Figure 1D&#8211;1E); for example, use a precut pierceable sealing film (see the Table of Materials). Ensure seal by rolling with a rubber roller. 
	NOTE: Pierceable silicone mats could potentially offer a reusable option, provided appropriate cleaning between uses, but the silicone mats that we tested easily split along the pierces and would not offer equal protection to any following plates.</li>
	<li>Place plate in refrigerator (4 &#176;C) to keep cool.</li>
</ol>
4. Transfer of sub-samples
NOTE: See step 4.13 for modifications when soil sample is very small.
<ol>
	<li>Place 24 of the 2 mL sub-samples into an ice block for cold storage.</li>
	<li>Using the sample name and well location sheet choose the correct 200 &#181;L flat-capped PCR tube.</li>
	<li>Take a 2 mL sub-sample tube and vortex for ~5 s to ensure homogenization.</li>
	<li>Using flame and bleach sterilized tools load ~200 &#181;L of the first sample into the correct flat-capped PCR tube (Figure 1F).</li>
	<li>Repeat steps 4.2&#8210;4.4 until all 24 samples have been loaded. Then move to step 4.6.</li>
	<li>Using a bleach-dipped paper wipe, clean the outside of the 200 &#181;L flat-capped PCR tube.</li>
	<li>Invert the tube and tap on the bench to move sample to top of the tube. With bleached and flame-sterilized scissors, clip the bottom of the PCR tube to create an opening for sample to fall into the 96-well plate (Figure 1G).</li>
	<li>Locate the correct well on a 96-well plate and pass the tube across plate with cut end facing up until reaching that well. Tilt the plate slightly to facilitate puncturing of precut pierceable sealing film, and only when tube is directly above the correct well carefully invert it so that cut tip fits into the well. Using sterilized tools, tap the top of the PCR tube until all soil has fallen from the tube into the well. Leave the tube within the well with lid closed (Figure 1H&#8211;1J).</li>
	<li>Remove one flat-capped 200 &#956;L PCR tube at a time and add 750 &#956;L of bead solution to that well&#160;(Figure 1K). Push the 200 &#956;L flat-capped PCR tube all the way down into the well and mark the top with sharpie to indicate that this well has been loaded. Repeat this for each sample.</li>
	<li>Once all 24 samples have been loaded and bead solution added, remove the 2 mL sub-sample tubes and replace with 24 unsampled tubes.&#160;</li>
	<li>Repeat steps 4.1&#8210;4.10 until all 95 wells have been loaded with sample or blank and bead solution.&#160;Remove tubes without passing them over open wells (Figure 1L).</li>
	<li>Carefully remove pierceable film and replace the rubber cover to protect the plate until beginning extraction (Figure 1M&#8211;1O).</li>
	<li>For very small quantities of soil that are also fine-grained, add 750 &#181;L of bead solution to the sample tube and use a wide-bore pipette tip to transfer all contents to the appropriate well. Place PCR tube into the opening to prevent double loading. 
	NOTE: Be careful when implementing this modification as particulate organic matter or small rock fragments larger than the bore of the pipette tip can clog the pipette and make transfer of the sample slurry difficult.</li>
	<li>As needed freeze at -20 &#176;C and store plates loaded with sample and bead solution until planned extraction.</li>
</ol>
5. Comparison of plate loading methods
NOTE: In order to verify our novel method for loading of 96-well DNA extraction plates, we divided a single 96-well plate into three sections. We used three different methods of plate loading to compare potential for unintentional loading of soil and cross contamination. The three methods we used were the methods outlined in McPherson et al.9, Qiagen&#8217;s default loading protocol10, and the protocol we outline in this publication.
<ol>
	<li>Loading soil into the plate
	<ol>
		<li>Section a 96-well plate into three four-column blocks. Load each block using one of the three method mentioned above.</li>
		<li>Load soil into every other well so that sample and blank wells are staggered. This arrangement allows for maximum opportunity for inadvertent sample loading and cross contamination.</li>
		<li>Freeze the 96-well plate at -20 &#176;C until DNA extraction.</li>
	</ol>
	</li>
	<li>DNA extraction
	<ol>
		<li>Extract DNA according to manufacturer&#8217;s 96-well plate extraction protocol10.</li>
		<li>Store the DNA extracts at -20 &#176;C until further analysis.</li>
	</ol>
	</li>
	<li>Quantification of DNA in blank wells
	<ol>
		<li>Thaw DNA extracts at room temperature, vortex and spin down using a plate spinner.</li>
		<li>Measure and record DNA concentrations of blank wells using a spectrophotometer.</li>
		<li>Use ANVOA and Tukey&#8217;s post-hoc test to analyze differences in mean DNA concentration in blank wells. 
		NOTE: Significant differences were reported at &#945; = 0.05.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Thompson, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+communal+catalogue+reveals+Earth's+multiscale+microbial+diversity.">A communal catalogue reveals Earth's multiscale microbial diversity.</a> Nature. 551 (7681), 457-463 (2017).</li><li>Davis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+yield+and+accuracy+for+DNA+extraction+in+microbiome+studies+with+variation+in+microbial+biomass.">Improved yield and accuracy for DNA extraction in microbiome studies with variation in microbial biomass.</a> BioTechniques. 66 (6), 285-289 (2019).</li><li>Marotz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triplicate+PCR+reactions+for+16S+rRNA+gene+amplicon+sequencing+are+unnecessary.">Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary.</a> BioTechniques. 67 (1), 29-32 (2019).</li><li>Romdhane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cover+crop+management+practices+rather+than+composition+of+cover+crop+mixtures+affect+bacterial+communities+in+no-till+agroecosystems.">Cover crop management practices rather than composition of cover crop mixtures affect bacterial communities in no-till agroecosystems.</a> Frontiers in Microbiology. 10, 1618 (2019).</li><li>Rochefort, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+environment+and+host+plant+genotype+on+the+structure+and+diversity+of+the+Brassica+napus+seed+microbiota.">Influence of environment and host plant genotype on the structure and diversity of the Brassica napus seed microbiota.</a> Phytobiomes Journal. 3 (4), 326-336 (2019).</li><li>Minich, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+and+understanding+well-to-well+contamination+in+microbiome+research.">Quantifying and understanding well-to-well contamination in microbiome research.</a> mSystems. 4 (4), 00186 (2019).</li><li>Walker, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lot+on+your+plate?+Well-to-well+contamination+as+an+additional+confounder+in+microbiome+sequence+analyses.">A lot on your plate? Well-to-well contamination as an additional confounder in microbiome sequence analyses.</a> mSystems. 4 (4), 00362 (2019).</li><li>Eisenhofer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contamination+in+low+microbial+biomass+microbiome+studies:+issues+and+recommendations.">Contamination in low microbial biomass microbiome studies: issues and recommendations.</a> Trends in Microbiology. 27 (2), 105-117 (2019).</li><li>McPherson, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+analysis+of+microbial+communities+in+soil,+rhizosphere,+and+roots+in+perennial+grass+Experiments.">Isolation and analysis of microbial communities in soil, rhizosphere, and roots in perennial grass Experiments.</a> Journal of Visualized Experiments. (137), e57932 (2018).</li><li>Qiagen, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> easy PowerSoil HTP 96 Kit Handbook. , (2019).</li></ol>

The authors report no conflicts of interest and have nothing to disclose.

This method reduces opportunities for well-to-well cross-contamination while loading high-throughput sample extraction plates and offers a more controlled means to load 96-well plates beyond existing plate loading strategies9,10. Contamination among wells can be more pervasive in 96-well plate extractions than in single-tube extractions, especially when automated6,7, and, though not specifically tested in any methodology, the risk of cross-contamination can be assumed highest when the wells of the extraction plates are left open for the duration of plate loading. Given the increased opportunity for cross-contamination in plate-based extractions compared to single-tube extractions, protecting wells with a sterile cover should be considered a key first step in loading samples into a 96-well plate. Despite this need, instructions provided in high-throughput extraction kits simply say to load the plate and to &#8220;avoid cross contamination between sample wells,&#8221; but do not provide any additional information as to how to do so10. To reduce contamination while adding samples to a 96-well extraction plate, McPherson et al.9 suggest covering the plate with cut strips of PCR sealing tape, peeling them back to access each well. This method reduces the total time that all wells are open, but wells are open for unequal amounts of time and some of the wells most susceptible to contamination&#8212;those nearest the sample&#160;being loaded&#8212;are exposed during the loading event. In addition, peeling of PCR sealing-tape has the potential to create static electricity which result in soil being transferred from well to well and could be responsible for the higher DNA concentrations in blank wells observed in our extraction. Furthermore, this methodology requires that samples are passed over uncovered wells before being placed in the well of choice, and nothing prevents a researcher from accidently loading two samples into a single well (double loading) or mixing up the location of two or more samples. If caught, double loading poses nothing more than a waste of resources. If, however, a double loading or mixing of sample locations goes unnoticed, experimental results may be distorted7.
The method we outlined above provides a controlled means to load high-throughput DNA extraction plates. Specifically, our method lowers the risk of cross contamination by keeping wells covered at all times. The wells are initially covered by sealing tape and are then kept covered by the PCR tube used for sample loading. Not only does this method reduce the risk of cross-contamination, but it also entirely prevents the risk of loading two samples into a single well. Once we load a sample into a well, the PCR tube serves as a block, preventing addition of another sample. By labeling all tubes with plate location (e.g., A1, A2... H11, H12) prior to loading samples, this method also provides a final check to ensure all samples have been loaded and no wells have been missed. While this method provides a more controlled means to load wells, anyone loading a plate should take extra care when passing a cut PCR tube over the plate. Even though the wells are sealed, any sample particles that fall onto the surface of the sealed plate could find their way into unintended wells when the technician pierces the film to load the correct samples into those wells. To avoid this issue, we tilt the 96-well plate to ~40&#176; prior to puncturing the sealing tape with the PCR tube. This approach helps to keep the open end of the tube upright and away from the incorrect wells.
When soil samples are of very small quantities and/or extremely fine-grained, using a wide-bore pipette tip instead of loading soil samples into PCR tubes may be more effective in transferring each sample. For this modification, the first buffer solution should be added to the sample tube and then both sample and solution pipetted into the 96-well plate. Adding a PCR tube to each well after this step will prevent double-loading. As mentioned in the protocol section, when using this modification, the researcher or technician should be very aware of particulate organic matter or small rock fragments as these have the potential to clog the bore of the pipette.
While no method of sample loading can entirely eliminate the risk of sample contamination, the method we outline above provides a means to reduce these risks substantially and entirely eliminate the risk of double loading and mixing of sample locations. Overall, we believe this method to be a great improvement on existing techniques for loading high-throughput DNA extraction plates and suggest that all microbiologists using high-throughput extraction plates move to using a method such as the one outlined above.

Recent advances in high-throughput sequencing of microbial communities are providing unparalleled sequencing depth and, consequently, an unprecedent glimpse into the functioning and diversity of Earth&#39;s microbiome1. As the ability to multiplex more and more samples onto a single sequencing lane increases, single tube DNA extraction has the potential to become a rate-limiting step in the generation of ecological data. However, new methods in high-throughput DNA extractions hold promise for processing large quantities of environmental samples with greater efficiency than has previously been possible2. These methods often involve using 96-well plates instead of single-tubes, thereby increasing the possible number of extractions that can occur simultaneously. As such, the practicality and efficiency of high-throughput extraction methods are evident and have been implemented for processing of environmental samples ranging from soil3,4 and plant tissues3,5 to human fecal matter2.
While these methods can dramatically speed along sample processing and DNA extraction, the initial step of loading soil and other ecological samples into 96-well plates is susceptible to cross-sample contamination. This type of well-to-well contamination can occur during DNA extractions6,7,8, and wells are particularly vulnerable in this first step before samples are suspended in a buffer solution. McPherson et al.9 demonstrate a method to load rhizosphere soils into 96-well plates using funnels and 8-well PCR strip covers, but while their method is a more controlled approach to loading plates, it still provides ample opportunity for contamination of neighboring wells when loading each sample. Additionally, the open wells allow the chance for a distracted researcher to place a sample into the incorrect well or add a sample into a well that already has been loaded. In addition, a variety of sample types prove to be ill-suited for loading with this method; wet samples often stick to the funnels and dry samples &#8216;jump&#8217; between wells due to static electricity.
To reduce opportunities for contamination among wells in the first step of high-throughput DNA extractions, we developed a new approach to loading soil samples into 96-well plates. Our methods both protect wells from environmental exposure and prevent us from accidentally loading multiple samples into one well (double-loading). We believe the method reported below offers promise to reduce the potential for contamination and as such provides a more controlled means to load 96-well plates for subsequent DNA extraction.

<table>
	<tbody>
		<tr>
			<td>18 oz WhirlPak</td>
			<td>Nasco</td>
			<td>B01065</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL centrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L micro-PCR tubes</td>
			<td>Thermo Scientific</td>
			<td>AB 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PowerSoil DNA extraction kit</td>
			<td>Qiagen</td>
			<td>12955-4</td>
			<td>We used a soil extraction kit but any 96-well format kit would work.</td>
		</tr>
		<tr>
			<td>Ice block for 2 mL centrifuge tubes</td>
			<td>Any</td>
			<td>Any</td>
			<td>Any ice block made for 2 mL tubes will work</td>
		</tr>
		<tr>
			<td>Ice block for 200 &#181;L micro-PCR tubes</td>
			<td>Any</td>
			<td>Any</td>
			<td>Any ice block made for 200 &#181;L tubes will work</td>
		</tr>
		<tr>
			<td>Micro scoopula</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Precut Pierceable Sealing Film</td>
			<td>Excel Scientific</td>
			<td>XPS25</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
	</tbody>
</table>

modified methods for loading of high-throughput dna extraction plates reduce potential for contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

This novel method was used successfully to load 96-well DNA extraction plates. Comparison of plate loading methods showed our method to have the lowest DNA concentration in the blank wells. The DNA concentration in the blank wells was significantly lower than the method proposed my McPherson et al.9 (p &#60; 0.05), though DNA concentrations in our method were not statistically different from the Qiagen default method (Figure 2). All three methods produced mean DNA concentrations under 2 ng/&#181;L, though only our new method &#160;produced wells with no measurable DNA concentration.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61405/61405fig01.jpg" /> 
Figure 1: Step by step methods to load samples into 96-well plates for DNA extraction while minimizing potential for cross-contamination among wells.&#160;Dark gray covers (present in steps A, B, N, and O) represent the silicon cover that comes with the extraction kit. The light gray cover (applied in step D, removed after step L) represents the pierceable PCR tape used to cover the plate while loading. Though not pictured, tools should be sterilized prior to loading each sample, and PCR tubes should be wiped with bleach prior to piercing the PCR tape. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61405/61405fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61405/61405fig02.jpg" /> 
Figure 2: DNA concentrations of blank wells.&#160;DNA concentration&#160;is represented in ng/&#181;L. Letters indicate significant pairwise differences in DNA concentrations at &#945; = 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61405/61405fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination at JoVE.com

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

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5.1K Views.  University of Wyoming. Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Video: Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

Summary

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Chapters in this video

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

High-throughput Saccharification Assay for Lignocellulosic Materials

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

Recovery of Adult Zebrafish Hearts for High-throughput Applications

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds