; Southern blot probe
Documents
Resources
Learning Center
Upload
Plans & pricing Sign in
Sign Out
Your Federal Quarterly Tax Payments are due April 15th Get Help Now >>

Southern blot probe

VIEWS: 52 PAGES: 7

  • pg 1
									                               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




                      IRES-LacZ
                        ~3.5kbp
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
               DNAs




                                 AhdI
            ~7kb                 ScaI
                                 SpeI
15kb           15kb
       miRNA
                     Predict Product Sizes




                         15kb                     15kb
                                          miRNA


                                                  ~7kb


• 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
                          bands

                          • 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