{"id":1272,"date":"2013-04-03T13:47:15","date_gmt":"2013-04-03T17:47:15","guid":{"rendered":"http:\/\/www.ambrosiasymbiosis.org\/labprotocols\/fungal-dna-extracton\/"},"modified":"2017-07-27T13:50:32","modified_gmt":"2017-07-27T17:50:32","slug":"fungal-dna-extracton","status":"publish","type":"post","link":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/fungal-dna-extracton\/","title":{"rendered":"Fungal DNA Extracton"},"content":{"rendered":"<h2>DNA Extraction From Fungal Plate<\/h2>\n<p>Please record all extractions correctly in the database. See database protocols for isolations for more information. Below is the current method:<\/p>\n<p>General Ex-N-Amp protocol:<\/p>\n<ol>\n<li>Prepare strip tubes with 40uL of Sigma Aldrich extraction solution. This can be found in aliquots in the shared box in the door of the small freezer.<\/li>\n<li>Scrape approximately 10uL of hyphae from fungal colony using a sterile pipette tip (20uL works best and is not used for much else) or scalpel.<\/li>\n<li>Add fungal material to extraction solution and vortex.<\/li>\n<li>Run Ex-N-A protocol on a thermocycler (96C for 30 minutes).<\/li>\n<li>Spin down with high rpm.<\/li>\n<li>Pipet off the top 25uL: that&#8217;s your extract. It is often safe to dilute this a bit if you will need more than 25 uL, but this is not standard procedure.<\/li>\n<\/ol>\n<p>Note: If BSA is needed, we have a stock of 2% molecular grade BSA in our freezer. This should be made into aliquots before using for samples. This is not standard, and only used in cases where it increases DNA amplification success.<\/p>\n<h2>OLD: DNA Extraction from mycangia<\/h2>\n<p>Modified Sigma Ex-N-Amp protocol:<\/p>\n<ol>\n<li>add contents of mycangium (if dissectable) or the body part with mycangium (for example, mandibular mycangium) to 10uL of XNA Extraction solution in a PCR tube<\/li>\n<li>mash the sample with a melted pipette tip<\/li>\n<li>run Ex-N-A protocol on a thermocycler (96C for 10 minutes)<\/li>\n<li>add 10uL of 3% BSA. DON&#8217;T ADD THE STANDARD DILUTION SOLUTION FROM SIGMA! It decreases yield sugnificantly.<\/li>\n<li>spin down with high rpm<\/li>\n<li>pipet off the top 10uL, and dilute with 10uL of water: that&#8217;s your extract<\/li>\n<\/ol>\n<p>&nbsp;<br \/>\n<!--\n\n\n<h2>Fungal CTAB extraction<\/h2>\n\n\n<b>NOTE<\/b>\n\n\n<ul>\n\t\n\n<li>Add 2% w\/v of PVP40 (polyvinilpyrrolidone) to the CTAB buffer. For example, to 10ml buffer add 0.2g PVP40.<\/li>\n\n\n\t\n\n<li>For really harsh extraction of DNA from fungi, add PVP 40, plus 1% beta-mercaptoethanol, and 3M sodium acetate (pH 5.2).<\/li>\n\n\n<\/ul>\n\n\n\n\n<ol>\n\t\n\n<li>Aliquot various size beads into 2 ml tubes (can use 1.5 ml, but the volume of beads has to be less than 0.7ml)<\/li>\n\n\n\t\n\n<li>add 500 uL CTAB into each tube<\/li>\n\n\n\t\n\n<li>add large piece of colony<\/li>\n\n\n\t\n\n<li>place in bead beater for 1 minute<\/li>\n\n\n\t\n\n<li>place on ice for 2.5 minutes<\/li>\n\n\n\t\n\n<li>repeat steps 4 and 5 two more times<\/li>\n\n\n\t\n\n<li>spin down briefly (1 min) to get rid of the foam<\/li>\n\n\n\t\n\n<li>add 500 uL chloroform, vortex well<\/li>\n\n\n\t\n\n<li>spin for 30 minutes full speed<\/li>\n\n\n\t\n\n<li>pipette off upper phase and combine with 500 uL freezer-cold isopropanol (may be stored overnight)<\/li>\n\n\n\t\n\n<li>spin 15 minutes at full speed<\/li>\n\n\n\t\n\n<li>pipette off alcohol. Dry pellet with low heat (~40C) in vacuum dryer.<\/li>\n\n\n\t\n\n<li>Dissolve pellet in 50uL water.<\/li>\n\n\n<\/ol>\n\n\n--><\/p>\n","protected":false},"excerpt":{"rendered":"<p>DNA Extraction From Fungal Plate Please record all extractions correctly in the database. See database protocols for isolations for more information. Below is the current method: General Ex-N-Amp protocol: Prepare strip tubes with 40uL of Sigma Aldrich extraction solution. This can be found in aliquots in the shared box in the door of the small [&hellip;]<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_s2mail":"yes","footnotes":""},"categories":[5],"tags":[],"class_list":["post-1272","post","type-post","status-publish","format-standard","hentry","category-fungi"],"_links":{"self":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1272","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/comments?post=1272"}],"version-history":[{"count":5,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1272\/revisions"}],"predecessor-version":[{"id":1974,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1272\/revisions\/1974"}],"wp:attachment":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/media?parent=1272"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/categories?post=1272"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/tags?post=1272"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}