Photo credit: Flickr user Caramosca (CC BY-NC 2.0) ; https://flic.kr/p/2bLPu4q
Tissue Collection for DNA Isolation
To extract DNA from Plodia, we use head, thorax, antennae, and legs as sample. Lysis step is the most important part of the isolation. Employing both enzymatic and physical lysis, between 400-700 ug of DNA yield can be extracted using spin column DNA isolation kit.
Rapid Genotyping (white gene)
Rapid genotyping foregoes the DNA isolation and washing steps. DNA from one leg is extracted out at high temperature into DNARelease solution. A fraction of that solution is then used as a template to amplify the desired DNA fragment using PCR. That fragment is then digested with the suitable genotyping restriction enzyme.
- One leg of a frozen Plodia individual is isolated with forceps while holding down the rest of the body using the lid of a microcentrifuge vial.
- Submerge the leg into 25 ul DNARelease solution in a PCR vial.
- Rinse forceps in 5% bleach, deionized water, and 70% Ethanol between samples. Wipe forceps with paper towel before handling the next sample.
- Heat the vials with legs and the solution at 98'C for 2 minutes.
- Take 1 ul of the sample is added into prepared PCR mix to amplify the gene of interest (in this example, white gene).
- Use Qubit to normalize the DNA amplicon concentration before restriction enzyme digestion. Nanodrop is not recommended since it cannot distinguish excess dNTP from amplicons.
- Digest PCR product with restriction enzyme (in this example, NlaV) and run on gel.