{"id":167,"date":"2017-04-19T15:17:31","date_gmt":"2017-04-19T19:17:31","guid":{"rendered":"https:\/\/health.uconn.edu\/mouse-genome-modification\/?page_id=167"},"modified":"2017-04-19T15:19:37","modified_gmt":"2017-04-19T19:19:37","slug":"bac-dna-for-injection","status":"publish","type":"page","link":"https:\/\/health.uconn.edu\/mouse-genome-modification\/protocols\/bac-dna-for-injection\/","title":{"rendered":"BAC DNA for Injection"},"content":{"rendered":"

Day 1<\/h2>\n

BAC containing bugs were grown in 250 mls of LB overnight.<\/p>\n

Day 2<\/h2>\n

BAC DNA was prepared using Qiagen\u2019s large construct kit. This kit includes an exonuclease digestion step that removes sheared BAC DNA and Bacterial DNA. The last prep yielded ~ 30 ug of BAC DNA as measured by a spec.<\/p>\n

Day 3<\/h2>\n

For constructs subcloned into BAC-Link vectors, use 10 ug of BAC and cut it with an I-PPO restriction enzyme (promega). Total volume of digest reaction should be ~ 200 to 250 ul.<\/p>\n

During digest, set up a 2 to 2.5 ml sepharose CL-4B column (Sigma CL4B200-100ml). Biorad poly-prep chromatography columns can be used to pour the sepharose.<\/p>\n

Equilibrate the column with injection buffer (10mM Tris pH7.5, 0.1mM EDTA, 100mM NaCl) ~ ten column volumes. Column flow rate is ~ 1ml\/three minutes so it will take 30 minutes to equilibrate the column.<\/p>\n

50 mls of injection buffer:<\/p>\n

    \n
  • 500 ul of 1M Tris pH 7.5<\/li>\n
  • 10 ul of 0.5 M EDTA pH 8.0<\/li>\n
  • 1 ml of 5 M NaCl<\/li>\n
  • 48.5 ml of water<\/li>\n<\/ul>\n

    Prepare 12 tubes for collection. Collect ~ 250 to 300 ul per fraction.<\/p>\n

    Digest for two to three hours then run digest through column.<\/p>\n

    Immediately start collecting fractions after sample is applied. After sample enters resin, gradually add injection buffer to column.<\/p>\n

    Measure DNA Concentration Using Fluorescent DNA Assay<\/h2>\n

    Using PicoGreen, carryout a DNA content assay on all the fractions. Run fractions on pulse field gel to verify optimal fraction.<\/p>\n

    In preparation for assay:<\/p>\n

      \n
    • Dilute DNA standard to 2 ug\/ml (6ul of 100 ug\/ml lamda DNA into 294ul TE). Then make three more consecutive 1\/10 dilutions to generate the following standards, 2 ug\/ml, 0.2 ug\/ml, .02 ug\/ml, .002 ug\/ml) (dilutions should be 30 ul into 270 ul of TE).<\/li>\n
    • Dilute Picogreen fluorescent substrate (20 ul into 4 ml of TE).<\/li>\n
    • Dilute your unknown fractions (20 ul into 180 ul TE).<\/li>\n
    • Pipet 100 ul of standards and unknowns into 96well plate in duplicate.<\/li>\n
    • Pipet 100 ul of substrate into each well.<\/li>\n
    • Measure on plate reader (Fluorocount).<\/li>\n<\/ul>\n

      Using linear regression, calculate concentrations of unknowns.<\/p>\n\n\n\n\n\n\n\n\n
      Concentration<\/strong><\/td>\nRFU<\/strong><\/td>\n\u00a0<\/strong><\/td>\nAVE<\/strong><\/td>\nDEV<\/strong><\/td>\nBlank Subtracted<\/strong><\/td>\n<\/tr>\n
      0<\/td>\n318<\/td>\n314<\/td>\n316<\/td>\n2.828427<\/td>\n0<\/td>\n<\/tr>\n
      1<\/td>\n421<\/td>\n377<\/td>\n399<\/td>\n31.1127<\/td>\n83<\/td>\n<\/tr>\n
      10<\/td>\n1028<\/td>\n983<\/td>\n1005.5<\/td>\n31.81981<\/td>\n689.5<\/td>\n<\/tr>\n
      100<\/td>\n6560<\/td>\n6792<\/td>\n6676<\/td>\n164.0488<\/td>\n6360<\/td>\n<\/tr>\n
      1000<\/td>\n56256<\/td>\n57600<\/td>\n56928<\/td>\n950.3515<\/td>\n56612<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

       <\/p>\n\n\n\n\n\n\n\n\n\n\n
      Unknowns<\/strong><\/td>\n\u00a0<\/strong><\/td>\n\u00a0<\/strong><\/td>\nAVE<\/strong><\/td>\nDEV<\/strong><\/td>\nCalculate Concentration of Unknowns<\/strong><\/td>\n<\/tr>\n
      1<\/td>\n356<\/td>\n328<\/td>\n342<\/td>\n19.79899<\/td>\n2.446066<\/td>\n<\/tr>\n
      2<\/td>\n337<\/td>\n317<\/td>\n327<\/td>\n14.14214<\/td>\n2.180382<\/td>\n<\/tr>\n
      3<\/td>\n5258<\/td>\n5171<\/td>\n5214.5<\/td>\n61.51829<\/td>\n88.74916<\/td>\n<\/tr>\n
      4<\/td>\n11509<\/td>\n11249<\/td>\n11379<\/td>\n183.8478<\/td>\n197.9365<\/td>\n<\/tr>\n
      5<\/td>\n12998<\/td>\n12997<\/td>\n12997.5<\/td>\n0.707107<\/td>\n226.6038<\/td>\n<\/tr>\n
      6<\/td>\n7355<\/td>\n7590<\/td>\n7472.5<\/td>\n166.1701<\/td>\n128.7435<\/td>\n<\/tr>\n
      7<\/td>\n2431<\/td>\n2457<\/td>\n2444<\/td>\n18.38478<\/td>\n39.67728<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"

      Day 1 BAC containing bugs were grown in 250 mls of LB overnight. Day 2 BAC DNA was prepared using Qiagen\u2019s large construct kit. This kit includes an exonuclease digestion step that removes sheared BAC DNA and Bacterial DNA. The last prep yielded ~ 30 ug of BAC DNA as measured by a spec. Day […]<\/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":"2026-04-27 00:04:10","action":"change-status","newStatus":"draft","terms":[],"taxonomy":""},"_links":{"self":[{"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/167"}],"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=167"}],"version-history":[{"count":3,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/167\/revisions"}],"predecessor-version":[{"id":170,"href":"https:\/\/health.uconn.edu\/mouse-genome-modification\/wp-json\/wp\/v2\/pages\/167\/revisions\/170"}],"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=167"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}