DNA sequencing: Difference between revisions
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The amount of mushroom you use is not critical, but smaller pieces often have a higher chance of working since there is less likelihood of contamination, and smaller pieces of mushroom allow you to use less DNA extraction reagent. I usually use about 20 milligrams, and if it is a tiny collection and I want to use less I use less DNA extraction solution. Since only 1 uL is needed for the template DNA, in theory you can get a good sequence from a piece of mushroom that is nearly too small to see. If the mushroom has gills, use that part because they have more DNA. If the mushroom is thick, break it in half and use part of the inside of the cap to reduce the chance of contamination. Get the mushroom piece with forceps and heat sterilize them between samples to reduce cross contamination. Flat tipped forceps may not need heat sterilization, you can just wipe them off. Sometimes I grind the samples with plastic pestles after adding extraction solution, but this isn't necessary and I am not sure if it helps. Grinding the samples probably increases the chance of success with something hard like a polypore but decreases the chance of success if your sample includes PCR inhibitors.
I suggest doing DNA extractions in .2 ml PCR tubes - that way you can incubate the DNA extractions in your thermocycler, and they are easier to store in the freezer. It is best to save DNA extractions so you can rerun PCR later with different primers to sequence additional genes.
PCR works at a wide range of DNA concentrations, and a likely cause of PCR failure is the presence of PCR inhibitors. More lengthy DNA extraction protocols can separate the DNA from PCR inhibitors. This is more often required with samples that have a lot of pigments.
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* 100 bp DNA Marker [http://www.the-odin.com/dna-ladder-100bp/ $10]
* Agarose [http://www.the-odin.com/agarose/ $10]
* DNA extraction supplies (NaOH and TRIS buffer, optionally plastic pestles. If you get pestles get the size for the .5 ml tubes so you can use 200 uL PCR tubes for DNA extractions) [https://www.ebay.com/sch/i.html?_nkw=Sodium+Hydroxide $10] [https://www.ebay.com/sch/i.html?_nkw=TRIS+base $10]
* PCR primers [http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/ $10]
* Gel loading dye [http://www.the-odin.com/6x-dual-color-dna-loading-dye/ $5]
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