How are you disrupting/grinding your samples? With just 200 microliters, these must be really small samples so you'll want to make sure you grind them really well. You can increase the proteinase K digest time & give the samples a quick flick to mix during the digest.

For PCR, I know it sounds counter-intuitive, but you can often get a higher success rate if you dilute out the DNA 1:10 or 1:100. That will dilute out PCR-inhibitors, but still leave more than enough DNA. I noticed you are only adding 1 microliter of DNA. It is challenging to use that tiny of a volume - easy to accidentally miss a sample, pipet onto the side & not in the reaction, etc. I'd recommend diluting the DNA so you can add a larger & easy to measure volume, say 5 microliters. Just reduce the volume of water in the PCR mix so the concentrations are correct.

That is a *really* fast PCR protocol. How small is your expected product? Is your positive control robust?

Good luck!

To improve DNA purity and PCR efficiency for beetle barcoding using Chelex 100, consider the following steps and recommendations: ### 1. Optimize DNA Extraction with Chelex 100 Chelex 100 is a chelating resin that can be used for rapid DNA extraction. It is particularly effective for small samples and can yield high-quality DNA suitable for PCR. #### Steps for Chelex 100 DNA Extraction: 1. **Sample Preparation**: Collect beetle specimens and store them in ethanol or another suitable preservative. 2. **Chelex 100 Solution**: Prepare a 5% Chelex 100 solution in distilled water. 3. **Sample Processing**: - Add a small piece of beetle tissue (e.g., leg) to a microcentrifuge tube containing the Chelex 100 solution. - Incubate the tube at 56°C for 30 minutes to 1 hour to facilitate cell lysis and DNA release. - Vortex the tube and then incubate it at 100°C for 8 minutes to denature proteins and stabilize the DNA. - Centrifuge the tube at high speed (e.g., 12,000 rpm) for 5 minutes to pellet the Chelex beads and cellular debris. - Carefully transfer the supernatant containing the DNA to a new tube, avoiding the Chelex beads. ### 2. Improve DNA Purity To ensure high purity of the extracted DNA, consider the following additional steps: 1. **Protein Precipitation**: - Add an equal volume of a protein precipitation solution (e.g., ammonium acetate) to the supernatant. - Mix gently and centrifuge at high speed to pellet the proteins. - Transfer the supernatant to a new tube, avoiding the protein pellet. 2. **DNA Cleanup**: - Use a commercial DNA cleanup kit to further purify the DNA if necessary. - Alternatively, perform a phenol-chloroform extraction followed by ethanol precipitation to remove contaminants. ### 3. Optimize PCR Conditions To improve PCR efficiency, optimize the PCR conditions and reagents: 1. **Primer Design**: - Use well-designed, specific primers for the target barcode region (e.g., COI for beetles). - Ensure the primers have appropriate melting temperatures and do not form secondary structures or dimers. 2. **PCR Reaction Components**: - Use high-quality Taq polymerase and fresh reagents. - Optimize the concentration of MgCl2, dNTPs, and primers in the PCR mix. 3. **PCR Cycling Conditions**: - Perform a gradient PCR to determine the optimal annealing temperature for your primers. - Use a hot start PCR to reduce non-specific amplification. - Adjust the number of cycles to ensure sufficient amplification without over-cycling. ### 4. Additional Tips - **Sample Storage**: Store beetle samples in ethanol at -20°C to preserve DNA integrity. - **Contamination Prevention**: Use sterile techniques throughout the DNA extraction and PCR setup to prevent contamination. - **Positive Controls**: Include positive controls with known DNA to verify the functionality of your PCR setup. ### References - The Chelex 100 method has been shown to be effective for DNA extraction in beetle barcoding studies, providing high-quality DNA suitable for PCR. - Optimizing the PCR conditions, including primer design and reaction components, is crucial for improving PCR efficiency. By following these steps and recommendations, you can improve the purity of your DNA extracts and enhance the efficiency of your PCR reactions for beetle barcoding.