No-boil Chelex 100 proteinase K genomic extraction.

I previously posted a Chelex 100 extraction protocol that while easy, cheap, and moderately effective calls for a boiling step to degrade the proteinase. The degradation step denatures template DNA causing it to become a single stranded product, which is unsuitable for genomic work and in my experience increases the difficulty of PCR.

A protocol published in Molecular Ecology Resources by Casquet et al. (2012) removes the boiling step. Their published protocol allows for high throughout and minimal cost. To their protocol I have added a grinding step which I believe increases DNA yield. I have successfully extracted and used DNA from just 1 leg of a microlepidopteran species.

 

Modified Chelex without boiling from Casquet et al. (2012)

  1. Add 10ul of protinease K (20 mg/ml) to each tube.
  2. Add 150ul of 10% Chelex 100 to each tube.
  3. Grind specimens with melted and sterilized pipet tips
  4. Incubate for 24 hours at 55 °C., swirling occasionally.
  5. Spin down to pellet the Chelex, pull DNA from top.

DNA extraction using Chelex 100 and proteinase K

Let me start by saying that the following extraction protocol is far from perfect. The product is dirty and often degrades at a faster rate than other extraction methods. Though for my work on insect genetics,  I find this protocol ideal because it is fast and cheap.

The extraction process consists of the addition of proteinase -K, Cheelex 100, and heat. Cheelex is used to protect the DNA from the degrading effects of proteinase K , which is added to free DNA from cells. Heat first speeds up the enzymatic degradation of the cells and then stops the enzymes by degradation. The extraction protocol is as follows:

  1. Prepare samples by placing tissues into 1.5 ml Eppendorf tubes, taking care to allow ethanol to evaporate (if the sample was stored in it).
  2. Add 200ul  of 10 % Chelex 100 per tube.
  3. Crush samples in Chelex 100 (I use melted pipette tips as tiny pestles).
  4. Add 1ul proteinase K (10 mg/ml) to each tube.
  5. Crush samples again.
  6. Vortex tubes for 10 seconds.
  7. Incubate on plate at 57 °C for 1 hour (up to 24 hours).
  8. Vortex for 10 seconds
  9. Boil at 95 °C for 5 minutes.
  10. Vortex for 10 Seconds
  11. Centrifuge at max speed (14,000 RPM) for 10 minutes.
  12. Take supernate and put in new labeled Eppendorf tubes.
  13. Create a working dilution (I usually do 1:10 or 1:50).
  14. Store at 4°C.Photo Oct 21, 10 33 26 AM

Evening Primroses

Many North American Mompha species feed on members of the evening primrose family, Onagraceae. Onagraceae is a cosmopolitian family with 17 genera and 655 species, with the center of diversity in the North American Southwest. This region is also a hot-spot for the largest genus within the evening primrose family, Oenothera; with 120 or so species.

For a master’s thesis, surveying Mompha diversity across the entirety of Onagraceae would be impossible because it would take years to complete! I have decided to instead focus my efforts on Oenothera that occur in the North American Southwest

Overlapping ranges, isolated populations, and multiple feeding niches within Oenothera will allow me to evaluate how host plants and biogeography have shaped speciation and distribution of associated Mompha.  

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Pictures of Oenothera Serrulata

Mompha

I am studying the diversification of a group of tiny moths called Mompha,  a genus within the family Momphidae. Mompha are moths with a ~2.5 to 8 mm forewing length, narrow-tufted forewings, and cryptic coloration. The only way to accurately  identify these moths to species are intensive genitalia dissections. There are roughly 40 described species of Mompha found in the United States with many species still unsubscribed because of the difficulty in their identification. Using molecular barcoding I can avoid complex genitalia dissections.  I hope to survey the diversity of Mompha associated with Oenothera, identify new putative species, and patterns of host plant mediated evolution.

mompha larvae

A picture of a Mompha larvae found in the fruit of an evening primrose

Photo Credit: Rick Overson landscapes of linalool flickr

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An example of an adult Mompha 

Photo Credit: Ken Childs from the Momphidae section at Butterflies and Moths of North America

Summer Collections

Assessing the diversity of Mompha associated with southwestern Oenothera was no small task. I traversed over 7000 miles! Thankfully I had the company and support of a fellow graduate student,  Ben. He was collecting leaf tissue from Oenothera section Calylophus as I collected Mompha from them.

We divided the journey up into three loops: (1) Texas, (2) Arizona/Utah/New Mexico, and (3) New Mexico/Oklahoma/Kansas. The trip took a little over a month and along the way we experienced brake failure, powerful monsoons, attacks from wild animals, and the great beauty of the southwest. I was able to have over 70 collection events which will lead to a strong dataset!