{"id":1580,"date":"2014-06-23T07:38:56","date_gmt":"2014-06-23T11:38:56","guid":{"rendered":"http:\/\/www.ambrosiasymbiosis.org\/labprotocols\/m13-amplification-procedure\/"},"modified":"2015-10-19T08:39:57","modified_gmt":"2015-10-19T12:39:57","slug":"m13-amplification-procedure","status":"publish","type":"post","link":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/m13-amplification-procedure\/","title":{"rendered":"M13 amplification procedure"},"content":{"rendered":"

PCR mastermix<\/b><\/p>\n\n\n\n\n\n\n\n
<\/td>\nper rxn<\/td>\n<\/tr>\n
Taq<\/td>\n\n

12.5 uL<\/p>\n<\/td>\n<\/tr>\n

M13<\/td>\n\n

2.5 uL<\/p>\n<\/td>\n<\/tr>\n

template<\/td>\n\n

1 uL<\/p>\n<\/td>\n<\/tr>\n

water<\/td>\n\n

9.5 uL<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

    \n
  1. Use the cycling conditions labeled as M13 (under Lab or Martin) on Hulcr lab thermocycler.<\/li>\n
  2. For extra care (ie publications), prepare a replicate PCR for each extraction.<\/li>\n<\/ol>\n

     <\/p>\n

    Running the gel<\/b><\/p>\n

     <\/p>\n

      \n
    1. Prepare a 1% agarose gel (careful, it will be fragile). Make sure the gel is level when pouring!<\/li>\n
    2. Use extra reagents, but make sure the volume will fit in the comb you use for making the wells:<\/li>\n<\/ol>\n\n\n\n\n\n
      ladder<\/td>\n9ul<\/td>\n<\/tr>\n
      template<\/td>\n9ul<\/td>\n<\/tr>\n
      dye<\/td>\n2ul<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

       <\/p>\n

        \n
      1. When loading, arrange samples from similar morphotypes together. If replicate PCRs were prepared, do not group them directly next to corresponding replicate sample.<\/li>\n
      2. Place both a 100bp and 1000 bp ladder on either side of every 5-10 reactions ran. This will aid in reading the gel, and will help to show if the DNA is moving at different speeds in different parts of the gel.<\/li>\n
      3. Run for up to 3 hours at 100-110V. Check every 45min-1 hour and take photo. The longer you run the gel, the more differentiated the bands will become, making it easier to read.<\/li>\n<\/ol>\n

         <\/p>\n

         <\/p>\n

        Reading the gel:<\/b><\/p>\n

        \u00a0<\/b><\/p>\n

          \n
        1. Use a photo editor (ie photoshop) to adjust the contrast\/brightness so that all bands can be seen and differentiated between samples.<\/li>\n
        2. Use a line tool in the photo editor to connect bands in ladders with the same lengths. Use the slope of these lines to gage the distances traveled between samples.<\/li>\n
        3. Note any differences in fragment location and presence\/absence between samples. Same species should be basically identical in their arrangement. Samples that appear different should be sequenced at a variable locus for confirmation.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

          PCR mastermix per rxn Taq 12.5 uL M13 2.5 uL template 1 uL water 9.5 uL   Use the cycling conditions labeled as M13 (under Lab or Martin) on Hulcr lab thermocycler. For extra care (ie publications), prepare a replicate PCR for each extraction.   Running the gel   Prepare a 1% agarose gel (careful, […]<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_s2mail":"yes","footnotes":""},"categories":[3],"tags":[],"class_list":["post-1580","post","type-post","status-publish","format-standard","hentry","category-general-molecular-work"],"_links":{"self":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1580","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\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/comments?post=1580"}],"version-history":[{"count":1,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1580\/revisions"}],"predecessor-version":[{"id":1820,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1580\/revisions\/1820"}],"wp:attachment":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/media?parent=1580"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/categories?post=1580"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/tags?post=1580"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}