{"id":181,"date":"2017-04-19T15:27:33","date_gmt":"2017-04-19T19:27:33","guid":{"rendered":"https:\/\/health.uconn.edu\/mouse-genome-modification\/?page_id=181"},"modified":"2017-04-19T15:27:33","modified_gmt":"2017-04-19T19:27:33","slug":"pcr-screening-for-targeted-stem-cell-colonies-in-96-well-plates","status":"publish","type":"page","link":"https:\/\/health.uconn.edu\/mouse-genome-modification\/protocols\/pcr-screening-for-targeted-stem-cell-colonies-in-96-well-plates\/","title":{"rendered":"PCR Screening for Targeted Stem Cell Colonies in 96 Well Plates"},"content":{"rendered":"
    \n
  • Use multiple channel pipet to pool DNA samples from each column, 10 \u00b5l from each well (one mixed sample contains DNA from eight wells). Mix thoroughly.<\/li>\n
  • Perform nested PCR.<\/li>\n<\/ul>\n\n\n\n\n\n\n\n\n\n\n\n
    \n

    PCR Mix<\/strong><\/p>\n<\/td>\n

    		\n

    Stock Concentration<\/strong><\/p>\n<\/td>\n

    		\n

    Amount (\u00b5l)<\/strong><\/p>\n<\/td>\n<\/tr>\n

    H2O<\/td>\n\u00a0<\/td>\n10.7<\/td>\n<\/tr>\n
    PCR Buffer<\/td>\n10 X<\/td>\n1.5<\/td>\n<\/tr>\n
    dNTP<\/td>\n2.5 mM<\/td>\n1.2<\/td>\n<\/tr>\n
    MgCl2<\/td>\n50 mM<\/td>\n0.45\u00a0<\/td>\n<\/tr>\n
    Primers<\/td>\n25 uM<\/td>\n0.2 each<\/td>\n<\/tr>\n
    Taq<\/td>\n5U\/ul<\/td>\n0.25<\/td>\n<\/tr>\n
    Total Volume<\/td>\n\u00a0<\/td>\n14.5<\/td>\n<\/tr>\n
    DNA Template<\/td>\n\u00a0<\/td>\n0.5<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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

    PCR Cycles<\/strong><\/p>\n<\/td>\n

    		\n
    \u00a0<\/h5>\n<\/td>\n
    		\n
    \u00a0<\/h5>\n<\/td>\n<\/tr>\n
    94\u00b0C<\/td>\n3 minutes<\/td>\n\u00a0<\/td>\n<\/tr>\n
    94\u00b0C<\/td>\n30 seconds<\/td>\n\u00a0<\/td>\n<\/tr>\n
    55\u00b0C<\/td>\n30 seconds<\/td>\n\u00a0<\/td>\n<\/tr>\n
    72\u00b0C<\/td>\n3 minutes (1 minute for 1kb)<\/td>\n35 cycles<\/td>\n<\/tr>\n
    72\u00b0C<\/td>\n10 minutes<\/td>\n\u00a0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

    Use the same nested PCR to screen individual well in the row that the pooled sample shows positive.<\/p>\n

    We use Platinum Taq (Invitrogen catalog number 10966) for PCR product < 3.5 kb. For PCR product longer than 3.5 kb, we use Elongase (Invitrogen catalog number 10480-010).<\/p>\n<\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"

    Use multiple channel pipet to pool DNA samples from each column, 10 \u00b5l from each well (one mixed sample contains DNA from eight wells). Mix thoroughly.Perform nested PCR.PCR MixStock ConcentrationAmount (\u00b5l)H2O\u00a010.7PCR Buffer10 X1.5dNTP2.5 mM1.2MgCl250 mM0.45\u00a0Primers25 uM0.2 eachTaq5U\/ul0.25Total Volume\u00a014.5DNA Template\u00a00.5\u00a0PCR Cycles\u00a0\u00a094\u00b0C3 minutes\u00a094\u00b0C30 seconds\u00a055\u00b0C30 seconds\u00a072\u00b0C3 minutes (1 minute for 1kb)35 cycles72\u00b0C10 minutes\u00a0Use the same nested PCR to […]<\/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":"2026-04-21 22:28:08","action":"change-status","newStatus":"draft","terms":[],"taxonomy":""},"_links":{"self":[{"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/181"}],"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=181"}],"version-history":[{"count":1,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/181\/revisions"}],"predecessor-version":[{"id":182,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/181\/revisions\/182"}],"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=181"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}