IGEM:metu/2009/Notebook/wound dressing/2009/08/20

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20.08.2009

1. We couldn’t prepare competent cell. We missed OD. We will try again.

2. We tried to digest EGF4 with Xba1 and Pst1, GFP with Spe1 and EcoR1. Just Ecor1 digested

3. We tried to digest KGF1 with Spe1, KGF2 with Pst1. They weren’t digested

4. We think because of lacking of BSA digestion didn’t happen.

5. We prepared new leader

  1 µl   leader
  1 µl   10X loading dye
  4 µl   dH2O

6. Electrophoresis order:

  1)Leader eski / leader yeni / GFP4 (24) control / EcoR1 / Spe1  / GFP (28) control / EcoR1  / Spe1
  2)EGF4(6) / Pst1(1)  /Pst1(2) / Xba1 / EGF4(11) / Pst1 / Pst1(2) / Xba1

7.The following amounts were added to microtubes.

 Monodigestion (fast);
 KGF1(AI2-1) ,   monodigestion:                            
               water  14.09 µl                                 
               Buffer  2 µl                                      
               DNA     2.91 µl                                     
               Spe1    1 µl    =20 µl
              
 KGF1(AI2-3) ,  monodigestion:                            
               water  14.13 µl                                 
               Buffer  2 µl                                      
               DNA     2.87 µl                                     
               Spe1    1 µl    =20 µl  
            
 KGF2(rbs1) ,  monodigestion:                            
               water  13.41µl                                 
               Buffer  2 µl                                      
               DNA     3.59 µl                                     
               Pst1    1 µl     =20 µl     
         
 KGF2(rbs2) ,  monodigestion:                            
               water  13.99 µl                                 
               Buffer  2 µl                                      
               DNA     3.01 µl                                     
               Pst1    1 µl     =20 µl       
       
 EGF1(06.08.09) ,  monodigestion:                            
               water  14.02 µl                                 
               Buffer  2 µl                                      
               DNA     2.98 µl                                     
               Spe1    1 µl    =20 µl    
          
 EGF1(11.08.09) ,  monodigestion: 
               water  14.89µl                                 
               Buffer  2 µl                                      
               DNA     2.11 µl                                     
               Spe1    1 µl    =20 µl               
 EGF2(rbs1) ,  monodigestion:                            
               water  13.41µl                                 
               Buffer  2 µl                                      
               DNA     3.59µl                                     
               Pst1    1 µl    =20 µl               
 EGF2(rbs2) ,  monodigestion:                            
               water  13.99 µl                                 
               Buffer  2 µl                                      
               DNA     3.01 µl                                     
               Pst1    1 µl    =20 µl               

8. Electrophoresis order:

  1)Leader  / rbs1 control / rbs1- / rbs1 / rbs2 control / rbs2- / rbs2 / leader
  2)EGF1 control (06.08.09) / EGF1 cut  / EGF1 control (11.08.09) / EGF1 cut / KGF1CONTROL (AI2-1) / AI2-1 cut / AI2-3 control  / AI2-3 cut