Portable PCR devices aren’t fun technology — they are about solving fundamental problems in molecular biology research. After years of centralized laboratory testing, we’re finally seeing instruments that address the critical limitations of traditional approaches.

Polymerase Chain Reaction (PCR) is an essential tool. Traditionally, the method has been confined to laboratory settings, requiring benchtop thermocyclers, dedicated workspace, and stable infrastructure. The development of portable PCR systems has changed this, allowing researchers to generate results outside conventional lab environments and at the point of sample collection.

Why Consider a Portable PCR System?

For researchers accustomed to working in fully equipped facilities, a compact, transportable thermocycler might not seem immediately necessary. However, conducting PCR outside of the lab offers several practical advantages:

Versatility Beyond Just PCR

Portable PCR systems are adaptable to a range of applications, from species identification to antimicrobial resistance screening. Some systems, like Bento Lab, integrate additional functions such as gel electrophoresis, providing a more complete molecular biology toolkit in a single, transportable unit. The ability to conduct multiple workflows in one device makes portable PCR especially useful for exploratory and field-based studies.

These systems also complement other sequencing and genotyping technologies. For example, portable PCR can enhance sample preparation for metagenomic analysis with sequencing platforms like MinION. Researchers can pre-screen samples before committing to resource-intensive sequencing workflows, improving efficiency and data quality in the process.

Considerations When Using a Portable PCR System

While portable PCR systems provide clear advantages, researchers should consider a few practical limitations:

Conclusion

Portable PCR systems are not just about convenience; they change the way molecular biology is conducted. By enabling researchers to analyze samples at the point of collection, they reduce delays, improve data integrity, and expand the reach of molecular research beyond the traditional laboratory. While they do not replace high-throughput laboratory processing, they provide an indispensable tool for field-based research, decentralized diagnostics, and time-sensitive workflows. Bento Lab, with its integrated features, offers a practical and versatile solution for researchers who need mobility without compromising functionality.

We are excited to see this new preprint focused on antimicrobial resistance (AMR).

The preprint features Bento Lab as part of a molecular detection toolbox for bacterial antibiotic resistance gene markers, with different workflows for a) well-equipped labs, b) equipment-poor labs, and c) portable uses with Bento Lab and lateral flow strips.

The molecular toolbox is reported in Vargas-Reyes et al. (2024) and was developed by a collaboration of scientists from Universidad Peruna de Ciencias Aplicadas, Lima, Peru, and the University of Camerino, Camiro, Italy.

The group wanted to address the global health problem of antimicrobial resistance (AMR), where the use of antibiotics plus environmental factors create a selective increase in bacterial strains resistant to antibiotic drugs — a big problem if any of these bacteria become involved in human or animal infections!

“The development of a simplified detection system for ARGs [antimicrobial resistance genes] is crucial for investigating AMR across a spectrum of contexts, from clinical settings to environmental and veterinary surveillance.”

This problem can be particularly notable in low- and middle-income countries where sanitation and hygiene standards may be limited and molecular laboratories can be less well-equipped to run surveillance and tracking programs.

To help address this problem, the team set out to develop a testing “toolbox” that could combine the sensitivity, accuracy, and speed of molecular techniques such as qPCR and DNA sequencing, with the low costs of more conventional antimicrobial susceptibility testing. They also wanted to allow their toolbox to be used in a wide range of test scenarios, from well-equipped laboratories to portable on-site applications.

“This toolbox is composed of a collection of primers, crRNAs, and well-defined protocols to be applied in laboratories with different equipment availability.”

The solution the team came up with was a set of workflows that first amplified specific antimicrobial target genes using conventional PCR, and then detected specific nucleic acid sequences of antimicrobial genes or plasmids using CRISPR technology. This combination of techniques is rapid, extremely sensitive and specific, and low-cost.

The CRISPR detection system involves a targeting sequence called crRNA that is synthesised to match the gene region of interest. When crRNA binds to the complementary target DNA:

The authors adapted this technique so it could be used in a range of lab conditions:

  1. In a high-equipped laboratory scenario, fluorescence can be detected and quantified precisely for large numbers of samples using a multimode microplate reader.
  2. In a less-well-equipped laboratory, fluorescence can be qualitatively detected using a blue light transilluminator
  3. In an on-site scenario, the Bento Lab portable PCR workstation can be used for PCR and CAS12a/crRNA incubations, and a Lateral Flow Assay platform (from Milenia Biotec GmbH) used to detect the cleaved reporter probe. In this scenario, the reporter probe contains biotin which (when the probe is cleaved by Cas12a) binds tightly to the streptavidin protein present in the lateral flow test (T) band. This allows the results to be visualised on the lateral flow strip by a colour change rather than by fluorescence.

Using these methods, the authors were able to detect less than 3x 10-7 ng of DNA or 100 colony-forming units per mL, detecting antibiotic-resistance genes conferring resistance to the antibiotics cefotaxime, other β lactams, and also amphenicols. Impressively, the assays’ sensitivity was 10x to 100x the lowest target concentration that could be detected using PCR and agarose gel electrophoresis analysis.

The workflows designed for different lab scenarios worked equally well in detecting the presence of two antimicrobial resistance genes in the authors’ test panels, although transilluminator-based detection and Lateral Flow Strip assays were only capable of qualitative, rather than quantitative, detection.

The authors say that their research “presents an optimized molecular detection toolbox for two of the most widespread genes conferring resistance to beta-lactams (blaCTX-M-15) and amphenicols (floR) and Int1 integrase, using the CRISPR-Cas technology.”

“The technical features of our toolbox are valuable because they enable accurate, consistent, and cost effective monitoring/surveillance efforts. These aspects are particularly beneficial for resource-limited settings lacking an adequate, cutting-edge, laboratory infrastructure.“

It’s a very cool toolbox of techniques, and it’s great to see Bento Lab was used as part of the proof-of-concept! It’s also a toolbox that should be useful for the detection of a wide range of other antibiotic resistance markers, as well as other markers, with minor modifications!

Congratulations to the team!

You can read the preprint here:

Vargas-Reyes et al. (2024). Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers. bioRxiv, 2024-11.

https://www.biorxiv.org/content/10.1101/2024.11.14.623642v1.abstract

Hey PCR enthusiasts!

We’re taking a break for the holidays, and we’ll be back in the New Year. We hope you have a great festive season!

This year our newsletter has radically changed. We started publishing weekly newsletter content on PCR-related methods and techniques, and also sharing regular additional content on X (formerly Twitter).

We’d love to hear from you. Do you like what we’ve been doing? What content do you want to see next year?

To close off the year, here’s a recap of the top ten most popular methods or articles we’ve shared this year:

⭐ 10. Direct PCR for microalgae

Did you know that you can use direct PCR for microalgae from liquid cultures? A study by Fei et al. (2020) indicates that you can! If the algae cultures are grown in salty marine media then you can centrifuge the algae and resuspend in distilled water to prevent PCR inhibition. This method could considerably simplify PCR-based study of these amazing and important microscopic organisms.

⭐ 9. Line-PCR, a rapid method of direct PCR sampling for plants

A new method by Lynagh et al. (2023) could make it much easier to do super-quick DNA sampling of plant leaves for direct PCR. The method, called “Line-PCR” by the authors, involves poking a hole in a leaf with a tiny piece of fishing line and putting the line straight into a PCR mix tube for direct PCR, or in water for later. Enough DNA is captured as a smear on the line to allow PCR for genotyping or other purposes.

⭐ 8. Single-tube nested PCR

Avoid PCR contamination issues when doing nested PCR by doing both rounds of PCR in the same tube. You can either dry the second set of primers on the PCR tube lids and shake to dissolve them for the second PCR (physical separation) (see Abath et al., 2002); or design the two primer sets so that the first set has a much higher annealing temperature (thermal separation) (you can find an open access example in Llop et al., 2000).

⭐ 7. Extracting PCR products from agarose gels using centrifugation

Extract PCR products from agarose gels for downstream purposes such as sequencing or ligation by using 0.5x TAE (Tris-Acetate-EDTA) buffer, cutting out the band, and centrifuging it through a filter. The liquid released should contain your DNA and have minimal PCR inhibitors. This method is very similar to the well known “freeze-and-squeeze” method, which uses a freezing step, but a study by Abraham et al. (2017) found that freezing beforehand isn’t necessary and direct PCR can be done provided 0.5x TAE buffer is used. As an extra suggestion, a low EDTA version of TAE buffer could be used to reduce PCR inhibition further, and the gel band could be centrifuged through a cut down filter pipette tip, making the method even easier.

⭐ 6. GelAnalyzer 23.1: Freeware electrophoresis gel analysis

Analyse your agarose electrophoresis gels and estimate band size and concentration using the excellent and free GelAnalyzer 21.3 software, available at www.gelanalyzer.com.

⭐ 5. Using laundry detergents for DNA extraction

Can you use laundry detergents for basic DNA extractions to save money? This idea has been successfully used for extractions of blood (Nasiri et al., 2005), bacteria (Mirnejad et al., 2012), and cattle hair (Guan et al., 2013), among others. But be careful if you want to make your methods reproducible by others (they may not be able to source a detergent brand, or the formulation may be changed over time), and don’t use detergents that contain DNAases as that would ruin your DNA extracts!

⭐ 4. Blocking primers to block specific unwanted DNA sequences

Did you know you can use modified primers to block the amplification of particular types of sequences (e.g. human or host DNA), to avoid your eDNA libraries being swamped by unwanted DNA? Two nice examples of this method are by Vestheim & Jarman (2008) for antarctic krill diet studies (blocking krill DNA), and by Bourret et al. (2021) for bird parasite metabarcoding (blocking bird DNA).

⭐ 3. Mu-DNA, a modular DNA extraction system

Extract DNA from anything and save money by using a homemade modular DNA extraction system that uses standard laboratory DNA extraction chemicals and reagents, called Mu-DNA (Modular Universal DNA Extraction Method), described by Sellers et al. (2018).

⭐ 2. Touchdown and Stepdown PCR

Improve the specificity and yield of your PCRs by using touchdown PCR. If your thermocycler is too basic to do this then you can use Stepdown PCR to approximate this method. You can read our resource on Touchdown and Stepdown PCR here.

⭐ 1. Booster PCR

Improve PCR specificity and yield using the same primer sets using this old method, which involves a first PCR with very dilute primers followed by a normal PCR with standard concentrations. You can read our thoughts on this topic here.

Thank you for joining us on our journey to make PCR available and accessible to anyone anywhere! If you’ve enjoyed our content, please let us know! And if you’d like us to cover anything in particular next year, please drop us a suggestion!

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Software and Applications
GelAnalyzer 23.1: Free Electrophoresis Gel Analysis Software

Electrophoresis gel analysis software can be useful to to improve estimates of DNA fragment size and concentration in electrophoresis gels. But what software can you use to do this?

One great option is GelAnalyzer 23.1, a freeware software application written and released by Dr. Istvan Lazar Jr. and Dr. Istvan Lazar Sr.

GelAnalyzer was first released in 2010 and since that time has been used in over 1K scientific publications according to Google Scholar.

Its core features include:

It’s free, works on Windows/Mac/Linux, it’s relatively easy to use, and it has a good walkthrough guide on the website, so why not give it a go?

You can find it at http://gelanalyzer.com, and the official reference is: GelAnalyzer 23.1 (available at http://gelanalyzer.com) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc.

Below is an example of GelAnalyzer’s interface in use, showing detection of calibration lanes (the DNA ladder), and estimation of the length of sample amplicons in comparison to the DNA ladder:

An example of amplicon molecular weight analysis in GelAnalyzer 23.1

Bento Lab Feature
Lab@Home vlog

Are you curious about setting up a PCR lab to work from home, and wondering what it would involve?

Take a look at our Lab@Home vlog series, where Dr. Jenny Shelton demonstrates DNA analysis on a kitchen table. 

In the series you find out what molecular biology at home REALLY looks like, and learn about core “PCR-at-home” skills, including how to consider sterility for a temporary lab on a kitchen table.

If you have ideas for experiments you want to see in the future, please let us know! We love making resources that enable anyone to do the PCR work they want to do, wherever they want to do it.

You can find all of our Lab@Home vlog series on the Bento Lab website here.

Dr. Jenny Shelton demonstrates DNA extraction from her kitchen table.

Methods and Techniques
Super-High Throughput DNA barcoding with ONT MinION

What’s the highest throughput and lowest cost for DNA barcoding that you can imagine being possible today?

In a new preprint, an amazing 100K invertebrate samples were sequenced in a single run using Oxford Nanopore MinION sequencing! The authors also demonstrated the sequencing of 10K and 2K samples using smaller and cheaper Flongle flow cells.

The authors, Hebert et al. (2023) from the Centre for Biodiversity Genomics, University of Guelph, Canada, examined COI DNA barcode recovery from 100K malaise-trapped arthropods, testing cost and effectiveness in batches of 2K, 10K, and 100K samples.

To keep costs down while sample numbers increased, the researchers used:

The authors compared their results to PacBio Sequel sequencing of the same extraction set, allowing the direct comparison of barcode production efficiency and sequence fidelity between the two different technologies.

Their barcoding workflow results across the three sample scales showed:

The authors calculate the sequencing costs at only $0.05/sequence at the 2K scale, and $0.01/sequence at the 100K scale. Importantly, this approach requires significantly smaller capital costs than other technologies, making it very well-suited for wide application around the world!

You can find the article preprint here:

Hebert et al. (2023). Barcode 100K Specimens: In a Single Nanopore Run. bioRxiv, 2023-11.

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Methods and Techniques
DNA Extraction Using Microwaves

Did you know that microwaving to burst open cells can be a useful method of DNA extraction, especially for cheap, rapid, and high-throughput workflows? It can be used as the main cell lysis process to produce a crude DNA extract for PCR, or as a step within a more complex extraction process to improve cell lysis.

As a cell lysis method, microwaving has the advantages of being rapid, inexpensive, high-throughput, single-tube, and capable of bursting cells without any chemicals that might impact downstream processes. It operates mostly by heating the sample to a high temperature, but mechanisms such as modification of cell membranes at lower temperatures have also been reported (for example bacterial cell membrane poration).

An early example was published by Goodwin & Lee (1993), where the authors tested it prior to a CTAB/chloroform extraction on a wide range of taxa across different kingdoms of life.

Since then, researchers have regularly published variations or instances of this method for specific applications, often omitting a CTAB/chloroform step and using direct PCR. Below are a few examples:

⭐Human samples: Taglia et al. (2022) describe the use of microwave DNA extraction for human samples (saliva, blood, semen) for forensics.

⭐Plant samples: von Post et al. (2003) describe a high-throughput DNA extraction from barley seeds for genotyping, using microwaving in an alkaline buffer followed by neutralisation with a Tris-HCl buffer, allowing the processing of thousands of seeds per day.

⭐Fungal samples: Ferreira et al. (1996) used microwaves to pop open dried fungal spores prior to PCR.

⭐Animal samples: Firmansya et al. (2023) compared three DNA extraction methods for mosquitos and found microwaving to produce comparable results to other methods while being a much quicker and cheaper workflow.

There are of course many other examples!

Do you use microwaving as part of your DNA extractions? If you do, we’d love to hear about how it works for you, and what your favourite methods are!

Bento Lab Pro Feature
Touchdown PCR

Do you have difficulty with finding the right annealing temperature for your PCRs, or have trouble with non-specific amplification? If you do, touchdown PCR (available with Bento Lab Pro) may be a good PCR method to try.

Touchdown PCR is most useful when very high levels of both specificity and yield are needed; when the optimal annealing temperature is unknown; or where a mixed sample may contain multiple targets that have different annealing temperatures for PCR. It uses annealing temperatures that start high but decrease by a small increment each cycle.

If you are interested in finding out more, we have an interesting short post exploring thoughts about Touchdown and Stepdown PCR on our website.

You can add a touchdown cycle to your PCR program on Bento Lab Pro following the instructions in the Bento Lab manual to program touchdown into a PCR step.

You can find out more about Bento Lab Pro on our website.

Are there any features you would like to see in future versions of Bento Lab? If there are, please drop us a message!

Methods and Techniques
Rapid Extraction of Very High Molecular Weight Plant DNA

For anyone interested in extracting very high molecular weight plant genomic DNA, for example for genome sequencing using long-read technologies such as Oxford Nanopore or PacBio sequencing, here is a rapid (compared to similar methods), simple, and economical method suitable for single-molecule sequencing, described by Li et al. (2020).

We found it particularly interesting because the extraction involves a careful, multi-step removal of cell walls, plastid DNA, and nuclei membranes, with wash stages after every step, rather than a single DNA extraction step followed by cleaning.

The extraction first removes cell walls to release intact nuclei using an osmotic buffer. Nuclei are then filtered, washed, and centrifuged before being gently lysed during a CTAB/chloroform extraction. Chloroplasts and plastid DNA are removed during these processes. The multiple wash steps help remove unwanted compounds such as secondary metabolites and chlorophyll, while the gentle lysis and buffer minimises DNA fragmentation.

Importantly, the method uses a novel extraction buffer containing Tris-HCl,  sucrose, spermidine trichlorohydride, and spermine tetrachloride. Spermine and spermidine are polyamines that stabilise and protect DNA in eukaryotic cells, absorbing free radicals and condensing DNA, allowing DNA extraction with minimal fragmentation.

Another critical step is to select young fresh leaves that are kept in the dark for up to 48 hrs prior to extraction to minimise the buildup of photosynthetic byproducts.

Using this method in cotton, blackgrass and strawberry, the authors were able to extract 100 micrograms of gDNA per 10 grams fresh tissue, with the majority of DNA extracted between 100 kb and 1000 kb in size!

You can find the article here:

Li et al. (2020). A simple plant high-molecular-weight DNA extraction method suitable for single-molecule technologies. Plant methods, 16(1), 1-6.

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Methods and Techniques
Line-PCR For High-Throughput Plant Genotyping

Line-PCR, developed by Lynagh et al (2023), is a rapid and cost-effective method of direct PCR for plant leaves that uses a minimal amount of plastic per sample.  It involves poking a hole in a fresh leaf with a 1–5 mm piece of fishing line and putting the line fragment directly into a PCR. While poking the leaf, just enough DNA is captured as a smear on the plastic surface to use for PCR, but not so much leaf tissue that it contains too many PCR inhibitors.

Line-PCR has been shown to work well with normal PCR master mix (with no modifications), for Arabidopsis, rice, potato, and false flax (Camelina sativa). It’s sufficiently quick and easy that large numbers of plants can be sampled in the greenhouse or the field in one session, making it ideal for high-throughput genotyping. The line can also be stored in sterile distilled water for up to 24 hrs prior to PCR with no observed lack of sensitivity, allowing PCR master mix and primers to be added when back in the lab.

In the same study, the authors also developed a similar method called “CutTip” where pipette tips are used instead of fishing line, and the end of the tip is cut off into a PCR tube. This method is not quite as easy or efficient but could be done if no fishing line is available at time of sampling.

As with all direct PCR methods no DNA extract is retained for repeats or different loci, and this should be taken into account if a DNA extract is needed.

The article, and videos of Line-PCR in action (in the supplementary data), can be found here:

Lynagh et al. (2023). Accurate Direct PCR with Arabidopsis and rice. Plant and Cell Physiology, 64(1), 1-3.

Product Highlight
Bento Lab Carry Case

Taking your Bento Lab on the road? Get peace of mind on the move with our durable carrying case!

Built tough to military-grade specifications, the case shields against water and dust, ensuring your lab stays safe even if it is briefly immersed in water. 

We stock two sizes: one snugly fits your Bento Lab, while the larger option can fit extra equipment like pipettes so you can keep everything organized for easy travel.

You can find the Bento Lab carry case in our online store here.

If you have any other great products you’d like us to stock, please drop us a message!

Methods and Techniques
Rapid MinION Workflow for Antimicrobial Resistance Gene Detection using Bento Lab

Bloemen et al. (2023) recently developed and optimised a three-hour workflow that allows DNA extraction and Oxford Nanopore library preparation for bacterial metagenomics for portable on-site use. The authors developed the workflow to detect antimicrobial-resistant genes present in chicken fecal samples as a test case, but they believe it could also be used for other complex sample types.

Their final portable on-site method (illustrated below by the authors) involved:

Overview of metagenomic workflows by Bloemen et al. (2023), CC BY 4.0 the authors.

The method returned comparable amounts of high-purity DNA compared to their laboratory-based workflow involving enzymatic lysis and Quick-DNA HMW magbead extraction, albeit with shorter fragment sizes (27 kbp vs 58 kbp).

Importantly, the authors also used a defined mock bacterial community to spike some of their samples. This allowed them to determine the effectiveness of different extraction steps (for example enzymatic lysis vs beat-beating), and to identify the strengths and weaknesses of the different DNA extraction treatments in terms of bacterial groups detected.

An interesting tweak was to reduce the Omnilyser X bead-beater battery voltage from 6 V to 1.5 V, providing a much gentler beat-beating action that doubled the fragment length of extracted DNA. While this did reduce DNA yield by nearly 50% it significantly improved the sequencing results.

You can find the article here:

Bloemen et al. (2023). Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance. Scientific Reports, 13(1), 19656.

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Methods and Techniques
DNA Extraction from Microorganisms via Hot Alkaline Fracturing of Cell Walls

Microorganisms with tough cell walls can offer challenges for DNA extraction. However, Liu et al. (2022) propose a simple, affordable, and rapid method that leaves DNA intact but available for PCR within damaged and fractured cells. The cells can then be pelleted, resuspended, and used directly for the amplification of DNA fragments of up to 6,500 bp and possibly more!

The authors call the method “PBC” (potassium hydroxide + boiling + centrifugation). It involves:

The PBC method is similar to other hot alkaline lysis methods, but it uses KOH rather than other alkalis, has a higher lysis temperature (100 °C vs 94 °C or lower), and uses suspended cell sediment as a DNA source rather than free DNA released into the extraction buffer.

The authors think that PBC extractions could be easily scaled up for routine processing of large numbers of samples, and had already tested it on hundreds of PCRs from smut fungus clones at the time of publication. However, it does rely on the use of a centrifuge which can be a limiting factor in throughput.

The study is a very detailed investigation with lots of extra detail, so well worth a read! You can find the article here:

Liu et al. (2022). A simple and rapid technique of template preparation for PCR. Frontiers in Microbiology, 13, 1024827.

Product Highlight
Agarose Tablets

Agarose tablets are a great way of making agarose gels for gel electrophoresis. No weighing, no spills, no waste, and consistent gel concentrations. They make gel preparation fast and hassle-free!

For a 1% gel, just drop a 0.5g tablet into 50 mL of TBE or other electrophoresis buffer. The tablets take a few minutes to disintegrate in the buffer, and you can then melt them using a microwave or other heat source (e.g. a rice cooker or camping stove) until they are fully dissolved and molten. 

This ease of use is particularly useful in educational settings, fieldwork, or for those new to electrophoresis.

If you want to try agarose tablets, we stock them in small quantities of 10 for $5.99 ($0.59 a gel), or you can buy a box of 200 for $72.99 ($0.37 a gel). You can find them in our store here.

If you have any other great products you’d like us to stock, please drop us a message!

Articles We Love
Mass Forensic Genotyping of Illegally Traded Pangolins

PCR and sequencing-based methods are essential tools in the identification and tracing of illegally traded animal or plant products, but large seizures can prove challenging and costly to process for provenance-tracing and prosecution.

A new approach, by Yeo et al. (2023), used Oxford Nanopore sequencing to significantly increase capacity while reducing costs, in the (probably) first example of high-throughput rapid genotyping and provenance tracing for wildlife forensic genotyping. The authors were able to DNA barcode an impressive 2346 scales from a large seizure of pangolin skins, and process them at a cost of less than 1 USD per sequence!

The authors used several optimisations to keep the cost down and to increase throughput, including:

Once the scales were sequenced and analysed, Yeo et al. (2023) were able to identify the scales to species and distinct haplotype level, matching the haplotypes to known phylogeographical populations.

The data indicated poaching primarily from Western-central Africa, Western Africa, and Gabon. The number and diversity of haplotypes poached indicated that skins were being centrally pooled before being shipped overseas. The scale of collections also indicated that continued intensive poaching may also impact the viability of local populations and create population bottlenecks.

Importantly, the methods developed and demonstrated by the authors represent a rapid, scalable, and cost-efficient genotyping framework that can be adapted for any large seizure of wildlife products.

You can find the article here:

Yeo et al. (2023). Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales. Conservation Biology, e14162.

The methods shown here could also be done on a smaller scale using Bento Lab, using a 32 well rather than 96 well plate format. This could make rapid-response genotyping and provenance tracing for wildlife forensics even more portable and adaptable!

We would love to hear from anyone interested in (or already doing) this kind of work!

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Methods and Techniques
Fast 2-Step PCR (VPCR)

Do you need faster PCR for detecting DNA targets using standard PCR equipment and polymerases? If you do, you may be interested in a two-temperature PCR method that could reduce PCR run times to as little as 30 minutes or even 8 minutes!

The method by Chen et al. (2019) is called “VPCR” (not to be confused with V-PCR or “viability PCR”), based on the “V-shape” of the thermal cycling program. It involves thermal cycling rapidly between two temperatures rather than three, with zero hold time durations. 

Temperature profiles of a conventional 3-step PCR program (denaturing, annealing, and extension) (left), and a V-shaped 2-step PCR program (heating and cooling) (right).

The denaturing, annealing, and extension steps occur during the dynamic heating and cooling processes instead of during temperature hold steps. Further time savings can be produced by also lowering the denaturing temperature and raising the annealing/extension temperature as amplicon concentration increases during the cycling.

Chen et al. (2019) found that VPCR could amplify a 500 bp amplicon in 16 minutes using a “fast” polymerase (KAPPA2G Robust), and in 32 minutes using a standard Taq polymerase. Shorter fragments amplified even more easily, and the authors managed to decrease the required time to amplify a 98 bp fragment to only 8 minutes!

The authors claimed no compromise in amplification efficiency or specificity for shorter fragments, which also means the method can be used for quantitative PCR (qPCR).

There are some disclaimers, in that success will probably vary depending on the specifics of the PCR application; the method may be best suited to shorter amplicons; and it may be easier to optimise for routine genotyping with standardised DNA extractions. We also haven’t yet tested this method ourselves.

Also, for use with Bento Lab, you will be limited by the shortest programmable temperature step being 5 seconds, which sets a minimum PCR time of around 25 minutes. However, this may be advantageous to some applications, and it’s still an amazing time saving if it works for your application.

You can find the article here:

Chen et al. (2019). Polymerase chain reaction using “V” shape thermal cycling program. Theranostics, 9(6), 1572.

Further examples of this method can be found in a later study of Pinelliae rhizoma (a chinese herb) by the same authors, and in a rapid genotyping study of tomato by a different group (subscription access only).

If you try VPCR, or are already doing something similar to it, and would like to share your experiences, please let us know!.

Product Highlight
Dipstick DNA Extraction Kit

If you want to rapidly extract and clean DNA for PCR from a wide range of tissues, then you may be interested in the ingenious filter paper dipstick method developed by Zou et al. (2020). It’s a great option for beginners, field-work, and low-throughput DNA extraction at an affordable price and using safe reagents.

You can rapidly extract PCR-quality DNA from a wide range of tissues in only 3 steps. The method involves grinding tissue in a lysis buffer, binding of DNA onto a filter paper dipstick, a wash step, and direct transfer of DNA into a PCR mix, all in 30 seconds!

We love this method because it’s very effective for some difficult tissue types, including fungi, lichens, and plants; and because it’s an easy method for anyone to do whether they’re beginners or more experienced.

Manufacturing dipsticks and solutions yourself can be difficult and time-consuming, so we’re very happy to be able to offer our Dipstick DNA Extraction Kit, which includes enough dipsticks and buffers for up to 100 extractions for $38.50. You can find it in our store here.

Methods and Techniques
Nucleic Acid Preservation Buffer (NAP Buffer or homemade “RNALater”)

Did you know that you can make your own DNA and RNA preservation solutions at a considerably lower cost than commercial solutions? Here is one recipe for a cheap but effective DNA and RNA preservative solution for animal tissues, their associated microbiomes, and possibly other tissue and DNA sources as well!

The solution has been called “Nucleic Acid Preservation” (NAP) buffer, and it appears to be based on the same chemistry as the popular RNALater™ preservation solution. To make 1 L all you need 7.44 g of EDTA, 7.35 g of sodium citrate tri-sodium salt dihydrate, and 700 g of ammonium sulphate in 1 L of water, adjusted to pH 5.2 using sulphuric acid. The method is more fully described in the article below:

Camacho‐Sanchez, M., Burraco, P., Gomez‐Mestre, I., & Leonard, J. A. (2013). Preservation of RNA and DNA from mammal samples under field conditions. Molecular ecology resources, 13(4), 663-673.

The authors reported that samples stored in NAP at ambient temperatures contained high-quality DNA for up to 10 months, and tissue DNA concentrations were significantly higher in NAP than in tissues stored in 95% ethanol or cryopreserved. RNA losses were higher but there was no significant difference between the performance of NAP and a commercial RNA preservation solution (RNALater™).

The solution appears to be relatively rarely cited compared to commercial alternatives (e.g. RNALater), but it has been used for quite a few diverse applications, including sheep dung microbiome studies, chicken gut microbiome metagenomics, equine faecal microbiota, preserving deer organs for virus detection, and tree bark biofilm metabarcoding, 

The chemistry is very similar or identical to that of RNALater, and you can find the now expired RNALater patent here: https://patents.google.com/patent/EP1657313A2/en.

The chemicals needed to make NAP buffer are inexpensive, safe, and widely available, except for the sulphuric acid for pH adjustment. And it seems quite simple to make if you have the chemicals, a pH meter, and some sulphuric acid to adjust the pH. So, if you need a cheap DNA and RNA preservative solution, then maybe this is one to try out!

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Methods and Techniques
Using Blocking Primers to Block Unwanted DNA Targets

Did you know that you could use modified primers (“blocking primers”) to block PCR for specific unwanted DNA targets, for example human DNA in eDNA surveys?

Blocking primers are oligonucleotides (short single strands of DNA) that have been modified to prevent amplification after they bind to their target binding regions. One common modification is the addition of three carbon atoms at the 3’ end of the primer (a C3 spacer modification), but several other blocking modifications have also been developed. Blocking primers can be designed to be specific to a particular unwanted DNA target, such as human DNA in eDNA surveys, and used to block amplification of these targets by various mechanisms.

One blocking approach is to design annealing-blocking primers that are specific to the unwanted DNA but also overlap with the binding site of the normal primers. This means that they actively compete with the normal primers for the unwanted DNA templates and can prevent all or most of the normal primers annealing if used in a high enough concentration.

A second approach is to design specific blocking primers for a region downstream of the binding site of the normal primer, thereby preventing further extension by the polymerase during PCR.

Blocking primers are usually added in a very high concentration compared to normal primers (e.g. 10x the concentration) to ensure good competition for binding sites on unwanted DNA fragments.

A good illustration and explanation of the use of blocking primers can be found in a study by Vestheim & Jarman (2008), investigating the diet of Antarctic krill from DNA in krill stomachs, where blocking primers allowed the amplification of a wide range of algal DNA regions with almost no krill DNA detected. Another example is in a study by Bourret et al. (2021) involving the metabarcoding of bird parasites, where blocking primers allowed the amplification of many different parasites including helminths, ciliates, bacteria, and fungi.

Blocking primers are only sometimes the best solution, however, as they can impact what is detected beyond the intended blocked DNA. For example, Zhang et al. (2020) found blocking primers for human DNA also reduced the number of fish species detected during an eDNA survey.

Product Highlight
HOT FIREPol® Blend Master Mix Ready to Load

If your PCRs suffer from non-specific amplification during room temperature set-up and you can’t easily set reactions up on ice, you may want to consider using a hot-start mastermix that only activates when thermal cycling begins.

We recommend HOT FIREPol® Blend Master Mix Ready to Load! It’s an optimised ready-to-use PCR master mix for more demanding applications, containing:

It’s cost-effective at only $0.34 per 20 µL reaction (when bought in 1 mL volumes), and it’s ideal for both laboratory and fieldwork use because the mastermix is specifically designed to allow up to a month of room temperature storage while retaining full performance. We also sell it in smaller volumes that may be more convenient for smaller projects or fieldwork.

Software and Applications
Primer-BLAST

Primer-BLAST is a great free online tool that anyone wanting to design or test primers should know about.

It’s a very popular and highly cited web tool created at NCBI to help people design primers and check for specificity against publicly accessible DNA sequences held in GenBank.

A few great things about Primer-BLAST are that:

A note of caution however: designing good primers can be complicated, so it’s best to use Primer-BLAST as one of several primer design tools and not to rely on it exclusively. There may be better ways to get to the primers you need.

Other primer design methods include using multiple sequence alignments to visually spot sequence-specific motifs and polymorphisms, or designing primers around single nucleotide polymorphisms for PCR assays such as ARMS-PCR.

You can find out more about Primer-BLAST here:

Ye et al. (2012). Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC bioinformatics, 13, 1-11.

And you can find the web portal for Primer-BLAST here.

Hey PCR enthusiasts,

Here are some updates from Bento Lab!

Methods and Techniques
Cheap DNA Extractions Using Laundry Detergents

Can you use biological laundry detergents instead of laboratory grade detergents and proteases to make an extremely cheap and accessible DNA extraction method with no refrigeration requirements?

Here are a few examples of people who have made laundry detergent extractions a reality:

Other methods can also be found in the literature that involve chloroform or magnetic beads to clean the DNA further.

However, a few notes of caution:

Product Highlight
Bento HotSHOT DNA Extraction Kit

If you are looking for simple, affordable DNA extraction kits, we have a few DNA extraction kits.

You can rapidly extract PCR-quality DNA from a wide range of tissues with the HotSHOT method. This kit is a simple, two step protocol performed in a single PCR tube.

If you want to skip making the solutions yourself, you can get our HotSHOT DNA Extraction Kit, which includes enough reagents for up to 400 rxn for $36.50.

Papers We Love
BaseLess: DNA detection using MinION without basecalling

Field sequencing technologies using Oxford Nanopore MinION are extremely portable, but they still require a mid-to-high specification laptop for basecalling in the field. This is a large additional expense that could limit deployment in resource-limited scenarios.

To overcome this limitation, some researchers are working to develop direct detection of target DNA from raw MinION signals using trainable neural networks, that can run without basecalling on a $100 single-board computer.

A group of researchers aiming to achieve this goal have produced a pipeline called “baseLess”. BaseLess uses easily configurable and trainable neural network arrays to directly interpret the raw nanopore signal (called the “squiggle”) as DNA strands flow through the pores. It then directly matches squiggle signals to reference genomes.

A key priority was to make the neural network array quick and easy to configure without retraining, so it can be easily used to detect different DNA targets. Another priority was to make it very lightweight, so that it requires ~1 MB per species model rather than hundreds of MBs (as with current base-calling methods).

BaseLess is capable of identifying organisms in its training set given a target genome and off-target genomes, for example detecting one fish species out of a range of candidate species. It’s also able to pick out specific target amplicon sequences from mixtures of environmental DNA, for example potentially pathogenic bacteria from gut microbiomes.

Its authors say baseLess needs further development to outperform current basecalling methods. However, they believe that it “has more potential to be developed into an accurate yet flexible sequence detection tool than its competitors, especially after it receives further optimizations”, due to its rapid training methods and low computational overhead.

It’ll be very exciting to see how this technology develops in the near future!

You can find the article here:

Noordijk et al. (2023). baseLess: lightweight detection of sequences in raw MinION data. Bioinformatics Advances, 3(1), vbad017.