Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement

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Stem region replacement

    
      
        
	  
	  Start
	  
	

	  
	  
PCR

	  
	  
	    
	      
	        
		  
Fri Aug 18

		
	      
	      
pHN1257

	    
	  
	

	  
	  
Transformation

	  
	  
	    
	      
	        
		  
Tue Aug 26

		
	      
	      
R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice	2min

	    
	  
	

	  
	  
Digestion

	  
	  
	    
	      
	        
		  
Wed Sep 10

		
	      
	      
Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer)

	    
	  
	

	  
	  
Ligation

	  
	  
	    
	      
	        
		  
Wed Sep 10

		
	      
	      
Ligate R0010 with pHN1257

	    
	  
	

	  
	  
Transformation

	  
	  
	    
	      
	        
		  
Wed Sep 10

		
	      
	      
Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice	2min

	    
	  
	

	  
	  
Colony PCR

	  
	  
	    
	      
	        
		  
Thu Sep 11

		
	      
	      
R0010-stem-dT 100up f 162primer R(あとで書く)

	    
	  
	

	  
	  
mini-Prep

	  
	  
	    
	      
	        
		  
Fri Sep 12

		
	      
	      
R0010-stem-dT

	    
	  
	

	  
	  
PCR(confirm)

	  
	  
	    
	      
	        
		  
Fri Sep 12

		
	      
	      
R0010-stem-dT 100up 162 KOD FX NEO

	    
	  
	
	
      Complete!