# DNA extraction from beetles [EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab) Updated 1 July 2022 ![](https://i.imgur.com/M2oWUHL.png) ## Project goal For RADseq genotyping, we need DNA samples from individual beetles containing at least 50 μl of high molecular weight (>10kb) DNA at a concentration of 20 ng/μl. Final sample concentrations must be 20 ng/μl and normalized across samples. [Extracted samples catalogued here](https://docs.google.com/spreadsheets/d/1W2-Lrw9Lcm-Eyancy-ZuoRJ0lEqZ9F1WedBFYxIYl-s/edit?usp=sharing) Flour beetles are tough samples. They are small and contain relatively little DNA. Their exoskeletons are tough, and they contain aromatic chemicals that may inhibit DNA extraction. This protocol has been modified from the Qiagen DNeasy Blood & Tissue Kit protocol for optimal extractions from whole flour beetles by Devin O'Brien, Chloe Schiff and Dave Angelini. ## Protocol ### Day 1 #### Before starting - Be sure specimens are **photographed**, if necessary - Buffer AW1 and Buffer AW2 are supplied as concentrates. Before using them for the first time, add the appropriate amount of **ethanol** (96-100%) as indicated on the bottle to obtain a working solution. - **Preheat the incubator** with rocking platform to 56°C #### Procedure - **Thaw frozen tissue** at room temperature for 10 min - If specimens are stored in ethanol, **air dry on a KimWipe** for 1-2 min - Place the whole beetle in 180 µl of **Buffer ATL** and **homogenize** completely using a plastic pestle and power drill for at least **1 min**. - Add 20 µl **Proteinase K**. - Mix thoroughly by vortexing - **Incubate at 56°C** overnight on a tube rotator. ### Day 2 #### Before starting - **Preheat Buffer AE** to 56°C #### Procedure - **Vortex for 15 s** - Add 200 µl **Buffer AL** and immediately mix thoroughly by vortexing for 15 s - Add 200 µl **ethanol** (96-100%) and immediately mix thoroughly by vortexing for 15 s :::info Buffer AL and ethanol can be premixed and added together when processing multiple samples ::: - Pipet the mixture onto the DNeasy Mini **spin column** placed in a 2-ml collection tube (provided with the kit). - **Centrifuge** at 6000 x g (8000 rpm) for 1 min - **Discard flow-through and collection tube** (The flow-through is not compatible with bleach.) - Place the spin column in a **new collection tube** - Add 500 µl **Buffer AW1** - **Centrifuge** at 6000 x g (8000 rpm) for 1min - **Discard flow-through and collection tube** - Place the spin column in a **new collection tube** - Add 500 µl **Buffer AW2** - **Centrifuge at 20,000 x g (14,000 rpm)** to dry the silica matrix - **Discard the flow-through and collection tube** :::warning Remove spin column **carefully**, so that the column does not come into contact with the flow-through. This will result in the carryover of ethanol from earlier buffers, reducing yeild. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifugation for 1 min at 20,000 x g (14,000 rpm). ::: - Place the spin column in a clean, labeled **1.5-ml microcentrifuge tube** (from lab supplies) - Pipet 30 µl of **preheated buffer AE** (56°C) directly onto the silica matrix - **Incubate at room temperature for 10 min** - **Centrifuge** for 1 min at 6,000 x g (8,000 rpm) to elute - Pipet the elutant **back onto the silica matrix** of the same column - **Incubate at room temperature for 10 min** - **Centrifuge** for 1 min at 6,000 x g (8,000 rpm) to elute - Store DNA samples at **-20˚C** (in the freezer). :::warning Be sure tubes are **labeled** and that identifying information is in your notebook and on any shared online spreadsheets. ::: :::info At this point, samples can be stored or you can proceed to spec them. ::: #### Final sample prepation - Use the **NanoDrop spec** to measure the concentration of the DNA (ng/µl). Record this information in your notebook. Concentrations should average about 50 ng/µl, ranging from 20 to 140 ng/µl. - Also note the A~260~/A~280~ ratio for the sample. (Pure DNA should have an A~260~/A~280~ ratio > 1.8.) - Based on the initial concentration, adjust the sample volume to a **final contration of 20 ng/µl**. #### Quality control - **Confirm high molecular weight** of samples by comparison to a 1-kb DNA ladder on an 0.8% agarose gel. --- [EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)