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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, 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.
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'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 ‘jump’ 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.
1. Laboratory bench and tool preparation for loading a 96-well plate
2. Sub-sampling and sample preparation
3. Plate preparation
4. Transfer of sub-samples
NOTE: See step 4.13 for modifications when soil sample is very small.
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’s default loading protocol10, and the protocol we outline in this publication.
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 < 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 con...
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...
The authors report no conflicts of interest and have nothing to disclose.
This research was supported by the Microbial Ecology Collaborative with funding from NSF award #EPS-1655726.
Name | Company | Catalog Number | Comments |
18 oz WhirlPak | Nasco | B01065 | |
2 mL centrifuge tubes | Fisherbrand | 05-408-129 | |
200 µL micro-PCR tubes | Thermo Scientific | AB 2000 | |
96-well PowerSoil DNA extraction kit | Qiagen | 12955-4 | We used a soil extraction kit but any 96-well format kit would work. |
Ice block for 2 mL centrifuge tubes | Any | Any | Any ice block made for 2 mL tubes will work |
Ice block for 200 µL micro-PCR tubes | Any | Any | Any ice block made for 200 µL tubes will work |
Micro scoopula | Any | Any | |
Precut Pierceable Sealing Film | Excel Scientific | XPS25 | |
Spatula | Any | Any | |
Surgical scissors | Any | Any |
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