1 
             
                                   Southern Blotting Technique  
                                        (Southern Analysis) 
                                                    
                                              (NOTES) 
             
            There  are  three  blotting  techniques  routinely  used  in  molecular  biology  for 
            detection of a specific DNA, RNA and protein sequence. In DNA and RNA samples, 
            sequence of nucleotides is detected, while in protein samples, amino acid sequence 
            is detected through blotting technique.  
             
                                                    
                                                                                
                                                    
             
            Southern blotting is a method for detection of a specific DNA sequence (nucleotide 
            sequence) in DNA samples.  
             
                                                                         
              Edwin Southern  
               
              The  Southern  Blotting  Technique  was 
              named after its inventor, Edwin Southern. 
              He was a British biochemist who developed 
              this  technique  in  Edinburgh  (Scotland)  in 
              the 1970s.                                                            
                                                                  Edwin Southern 
             
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                   Procedure 
                    
                   High molecular-weight DNA strands (large-sized DNA strands) are cut into smaller 
                   fragments  using  Restriction  Endonucleases  (RE).  Then,  the  RE-digested  DNA 
                   fragments are subjected to Agarose gel electrophoresis in order to separate them 
                   according to size.  
                    
                                                                               
                                                       Steps in Agarose Gel Electrophoresis 
                                                                               
                                                                               
                                                                                                                              
                    
                   If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel 
                   may be treated with an acid (such as dilute HCl), which breaks the DNA into smaller 
                   pieces,  thus  allowing  more  efficient  transfer  from  the  gel  to  nitrocellulose 
                   membrane (discussed in the nest steps). 
                   The DNA gel is placed into an alkaline solution (NaOH) to denature the double-
                   stranded  DNA  fragments  into  single  strands.  The  denaturation  in  an  alkaline 
                   environment may improve binding of the negatively charged DNA to a positively 
                   charged nitrocellulose membrane (discussion in next steps). It will also help the 
                   hybridization  of  the  radioactive  probe  with  single  stranded  DNA  fragments 
                   (discussion in next steps). 
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Agarose gel (ready to use) 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
                                                                                                                                                                          
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Gel with separated DNA fragments  
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
                                                                                                                                                              
                                                                                                                                                             Transfer  of  Single  stranded  DNA  fragments  to  nitrocellulose  membrane  is 
                                                                                                                                                             facilitated using buffer solution or salt solution system, which is as follows: 
                                                                                                                                                              
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                       Southern Blotting                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
                                                                                                                                                                                                                                                                                                                                                                                                                                                                           (1st step)                                                                                                                                                                                                                                                                                                                                                                                                                                                   
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                Weight (0.75 kg)                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Filter paper towel 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Nitrocellulose membrane 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Gel 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Filter paper wick 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Salt solution or buffer 
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                   A piece of sponge (or filter paper wick) is placed in a tank containing the buffer or 
                   salt solution. Later, Agarose gel, carrying single stranded DNA fragments, is put on 
                   top of the sponge.  
                    
                   A sheet of nitrocellulose membrane (nitrocellulose filter) is placed on top of the 
                   gel. To ensure good and even contact between gel and membrane, pressure is 
                   applied  evenly  to  the  gel  by  placing  a  stack  of  filter  paper-towels  (absorbent 
                   material) and a weight (0.5 to 0.75 kg) on top of the membrane and gel.  
                   Buffer solution (or salt solution) is used to prevent drying of the gel and to transfer 
                   the  DNA  fragments  from  gel  to  nitrocellulose  membrane.  Buffer-transfer  by 
                   capillary action from a region of high water potential to a region of low water 
                   potential (usually facilitated by filter paper tissues) is then used to move the DNA 
                   from the gel on to the membrane; ion exchange interactions bind the DNA to the 
                   nitrocellulose membrane due to the negative charge of the DNA and positive 
                   charge of the membrane. 
                    
                   Positions of the DNA fragments on the nitrocellulose membrane are identical to 
                   their positions in the gel. 
                    
                                                                                
                                                             Southern Blotting Procedure