Southern blot probe by hcj


									                               Southern blot flowchart

                                             Digest genomic DNA & run on an agarose gel
                                             Denature the dsDNA into ssDNA
                                             Transfer onto a nylon membrane overnight
                QuickTime™ and a
      TIFF (Un compressed) decompressor
         are neede d to se e this picture.   Label the probe with P32
                                             Hybridization overnight
                                             Visualize on X-ray film

Southern blot for positively targeted ES cells:
- check for multiple random integrations
- include on the paper as supplementary data
Southern blot probe

Identify Restrictive Enzymes

               • should not cut the highlighted area
               • digest genomic DNAs into various sizes
               • must produce a product smaller than ~30kbs
               • have good enzyme activity to cut genomic

            ~7kb                 ScaI
15kb           15kb
                     Predict Product Sizes

                         15kb                     15kb


• example) miR-320
      enzyme - ScaI
      expected size = 7 + 2.5 = 9.5kb
• predict sizes for all positive clones
       Develop the Southern Blot

                          • 1 out of 35 lines shows double

                          • let-7c-2/7b  high percentage
                          chimeras have been produced
AhdI          ScaI
                          recently, but they will be sacrificed

                          • DTA negative selection marker
                          should theoretically eliminate
                          random integrations

                          • However, before releasing KO
                          mice to JAX, we will definitely have
                          to perform Southern blot
ScaI          SpeI
Future directions
 Southern blot for all positively targeted ES cell lines before releasing
 Pipeline additional ~80 constructs from CHORI

Thanks to…
   Chong Park
   Jean Liu
   Rachel Cameron
   Michael McManus
   McManus Lab

To top