In an April 2020 webinar, MicroGEM’s Rob Thompson introduced a simple protocol for extracting DNA from insects using a single tube, without ionic detergents, magnetic beads, columns or centrifugation. He used case studies featuring a range of insects including fruit flies, anopheles mosquitoes, and African whitefly, and presented qPCR data comparing enzymatic lysis with traditional spin-column methods.
For your convenience, we have transcribed the Q&A portion of the webinar here. Read more to discover the advantages of temperature-driven, enzymatic extractions that provide maximum DNA recovery, a simplified workflow, and significant reduction of plastic and chemical waste – all in minutes, not hours.
Learning outcomes include:
• The differences between chemical and enzymatic, heat-driven DNA extractions
• A method to extract DNA from small/scarce/challenging insect samples in less than 20 minutes
• Optimizing extractions for downstream applications including PCR, qPCR, whole genome amplification, and amplicon sequencing
This webinar was recorded live by MicroGEM on April 1, 2020.
Q&A with Rob
Q: Will this work on dried insects, like dried mosquitoes, perhaps with modifications?
A: For particularly dry or old samples you would want to make sure you homogenize the sample instead of using the intact insect to get the most DNA as possible.
Q: Will this work for ticks?
A: Yes, Polar Research published a study in 2015 focusing on tick-borne pathogens. The publication can be found on our website. Another study published last year showed a group in Texas that sampled about 200 ticks and used the MicroGEM method to extract DNA to investigate molecular markers to help with species identification.
Q: Can I scale up and down the reagents based on the size of the insect?
A: Yes, as shown earlier, we have groups using parts of insects like mosquito legs and they scale down reagents to 10ul. You could easily scale up to 100ul 200ul if you wanted to extract from a particularly large sample. You would just pipette in the reagents to a larger tube like a 1.5ml Eppendorf tube and use a 1.5ml
Q: How do I perform homogenization?
A: I would just follow your normal method for homogenization. Using a handheld homogenizing tool you could grind up the sample in the MicroGEM reagents. If you wanted to you could freeze the samples in liquid nitrogen and then grind them up.
One thing that you would want to consider is the fact that TDE does not remove downstream inhibitors from the mix. If you are using a homogenizing buffer that contains chemicals that could inhibit PCR, sequencing…. These chemicals will remain in the mix and therefore could cause issues when you run your analysis. So try not to homogenize in harsh chemicals.
Q: Is the extract compatible with every PCR reagents?
A: Yes, it has been compatible with all the reagents we have tried.
Q: Our particular challenge is working tiny beetles (Coleoptera). We use ProK but can’t get it to digest through the Chitin. Is your enzyme capable of doing this?
A: We are more aggressive on chitinous samples due to the elevated temperature of our prepGEM Enzyme extraction @ 75°C. Some customers also use entomological pins to put a tiny hole in each beetle body to aid with DNA recovery.
Q: Who distributes your equipment in the US?
A: We sell directly on our website. As well, Stellar Scientific is a US distributor.
Q: When will the slide be made available?
A: The slides are available on-demand.
Q: Who is your contact or a customer support person in the US? We use EZ1 automated Qiagen which is expensive and only does 14 at a time, so I am very interested in your machine and protocol.
A: Jeff Hickey, our product specialist in the US, can help. He’s at firstname.lastname@example.org.
Business Development and Product Specialist, MicroGEM
Rob Thompson provides technical, business, and product development expertise to MicroGEM’s commercial, engineering, and science teams, guiding product development and enhancement activities. Rob's background is in cellular and molecular medicine having attained his BSc Hons degree from the University of Bristol in the UK working on epigenetics and TET proteins.
After graduating, Rob moved to China to study Mandarin at Jiaotong University in Shanghai before joining the MicroGEM team, initially working on the development of MicroGEM's PDQeX Nucleic Acid Extractor. He now leverages his deep understanding of the company’s unique DNA and RNA extraction capabilities to expand and improve product offerings across a wide variety of research and commercial applications.