IGEM:metu/2009/Notebook/wound dressing/2009/08/19

From OpenWetWare
Jump to: navigation, search
Igem-logo-150px.png WOUND DRESSING Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

Wound dressing logo.jpg

19.08.2009

1. Transformation result : We saw very few on plates. (we think because of compotent Top 10 cells transformation wasn't succesful)

2. Isolation of Rbs, AI2-ind,LUXR-Rbs, TRH, pEGF-Lard1, pRGF-HiA and storages of pEGF-Lard1, pRGF-HiA were made.

3. CaCl2 and LB were prepared.

4. We made (fast) monodigestion for GFP, EGF4, BB11 than purification and again (fast) monodigestion. Just BB11 was cutted. Then we made gell extraction. We couldn’t be sure for GFP and EGF4.

5. Double digestions of GFP(digested), EGF4 (undigested)were made.

  For EGF3,         Prefix             Suffix
  GFP               EcoR1              Spe1
  EGF4              Xba1               Pst1
  BB11              EcoR1              Pst1

The following amounts were added to microtubes;

  GFP(28.07.09) ,  monodigestion(fast):                            
                  water  14.9 µl                                 
                  Buffer  2 µl                                      
                  DNA     2.1 µl                                     
                  EcoR1   1 µl =20 µl               
  EGF4(06.08.09) , monodigestion(fast):                        
                  water  14.5 µl                               
                  Buffer  2 µl                                    
                  DNA     2.5 µl                            
                  Xba1    1 µl =20 µl                  
  BB11, monodigestion(fast):                            
                  water  11.8 µl                                 
                  Buffer  2 µl                                      
                  DNA     5.2 µl                                    
                  Ecor1   1 µl =20 µl                  
	

After PCR purification;

  For GFP,  
         28 µl  solution
          2 µl  Spe1
          4 µl  buffer
          6 µl  water    =40 µl  
  For  EGF4,
          28 µl  solution
           2 µl  Pst1
           4 µl  buffer
           6 µl  water    =40 µl  
  For BB11,
         28 µl  solution
          2 µl  Pst1
          4 µl  buffer
          6 µl  water    =40 µl  

6. Electrophoresis order:

  1)Leader / GFP control / GFP / EGF4 control / EGF4 / BB11 control / BB11 / Leader	
  2)Leader / GFP / GFP / EGF4  / EGF4 / BB11 / BB11 / Leader
  3)Leader / GFP / GFP / GFP control / Leader / EGF control / EGF / EGF
	

7. Mono digestions ( 16 hours incubation );

      EGF4                     1/06.08.09                          2/11.08.09
           water                14.48 µl                            12.87 µl
           Buffer Tango          2 µl                                2 µl
           DNA                   2.52 µl                             4.13 µl
           Xba1                  1 µl       = 20 µl                  1 µl             =20 µl
                                
      GFP                      1/24.07.09                           2/28.07.09
           water                13.43 µl                             12.87 µl
           Buffer EcoR1          2 µl                                 2 µl
           DNA                   3.57 µl                              2.06 µl
           EcoR1                 1 µl      = 20 µl                    1 µl             =20 µl