{"id":188,"date":"2017-04-19T15:33:17","date_gmt":"2017-04-19T19:33:17","guid":{"rendered":"https:\/\/health.uconn.edu\/mouse-genome-modification\/?page_id=188"},"modified":"2017-04-19T15:36:16","modified_gmt":"2017-04-19T19:36:16","slug":"ear-punches-yolk-sacs-tail-dna-for-pcr","status":"publish","type":"page","link":"https:\/\/health.uconn.edu\/mouse-genome-modification\/protocols\/ear-punches-yolk-sacs-tail-dna-for-pcr\/","title":{"rendered":"Ear Punches, Yolk Sacs, Tail DNA for PCR"},"content":{"rendered":"
    \n
  • Dissect yolk sac and rinse thoroughly in PBS.<\/li>\n
  • Place yolk sac or ear punch into 100 to 300 \u00b5l ProtK buffer with non-ionic detergents.<\/li>\n
  • Incubate overnight at 55\u00b0C.<\/li>\n
  • Place 5 \u00b5l of the ProtK\u2019d material into a PCR tube with 30 \u00b5l dH20, overlay with two drops of mineral oil. Too much tissue can inhibit the PCR.<\/li>\n
  • Run PCR reaction at 95\u00b0C for ten minutes to inactivate ProtK.<\/li>\n
  • Bring the PCR machine to soak at your annealing temperature and add PCR mix 15 ul.<\/li>\n
  • Begin PCR cycles.<\/li>\n<\/ul>\n

    ProtK Buffer with Non-ionic Detergents<\/h2>\n\n\n\n\n\n\n\n\n\n\n
    Final Concentration<\/strong><\/td>\nFor 100 ml<\/strong><\/td>\n<\/tr>\n
    50 mM KCl<\/td>\n5 ml of 1 M stock<\/td>\n<\/tr>\n
    10 mM Tris.HCl (pH 8.3)<\/td>\n1 ml of 1 M stock<\/td>\n<\/tr>\n
    2.0 mM MgCl2<\/td>\n0.2 ml of 1 M stock<\/td>\n<\/tr>\n
    0.1 mg\/ml gelatin<\/td>\n10 ml 1% stock<\/td>\n<\/tr>\n
    0.45% Nonidet P-40<\/td>\n0.45 ml<\/td>\n<\/tr>\n
    0.45% Tween-20 l<\/td>\n0.45 ml<\/td>\n<\/tr>\n
    H2O<\/td>\n83 ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

    Autoclave solution, aliquot and store frozen. Thaw immediately before use and add 25 ul ProK 20mg\/ml stock solution per 1 ml of buffer.<\/p>\n\n\n\n\n\n\n\n\n\n
    PCR Mix - Stocks<\/strong><\/td>\nOne Reaction<\/strong><\/td>\nTen Reactions (Make for 11 Reactions)<\/strong><\/td>\n<\/tr>\n
    dNTP 2 mM<\/td>\n5 \u00b5l<\/td>\n55 \u00b5l<\/td>\n<\/tr>\n
    10x PCR Buffer<\/td>\n4.5 \u00b5l<\/td>\n49.5 \u00b5l<\/td>\n<\/tr>\n
    Primers 25 uM<\/td>\n1 \u00b5l each<\/td>\n11 \u00b5l each<\/td>\n<\/tr>\n
    Taq Polymerase<\/td>\n0.25 \u00b5l<\/td>\n2.75 \u00b5l<\/td>\n<\/tr>\n
    H2O<\/td>\n3.25 \u00b5l<\/td>\n35.75 \u00b5l<\/td>\n<\/tr>\n
    Total Volume<\/td>\n15 \u00b5l<\/td>\n165 \u00b5l<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"

    Dissect yolk sac and rinse thoroughly in PBS. Place yolk sac or ear punch into 100 to 300 \u00b5l ProtK buffer with non-ionic detergents. Incubate overnight at 55\u00b0C. Place 5 \u00b5l of the ProtK\u2019d material into a PCR tube with 30 \u00b5l dH20, overlay with two drops of mineral oil. Too much tissue can inhibit […]<\/p>\n","protected":false},"author":38,"featured_media":0,"parent":45,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"acf":[],"publishpress_future_action":{"enabled":false,"date":"2026-04-22 20:47:28","action":"change-status","newStatus":"draft","terms":[],"taxonomy":""},"_links":{"self":[{"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/188"}],"collection":[{"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/users\/38"}],"replies":[{"embeddable":true,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/comments?post=188"}],"version-history":[{"count":3,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/188\/revisions"}],"predecessor-version":[{"id":191,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/188\/revisions\/191"}],"up":[{"embeddable":true,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/45"}],"wp:attachment":[{"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/media?parent=188"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}