Team:Tsinghua/Notebook

From 2008.igem.org

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PCR  
PCR  
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PCR by Pyrobest DNA polymerase
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{| border="1"
-
 
+
|+PCR by Pyrobest DNA polymerase
-
Reagent             Concentration/Activity       50ul    100ul
+
|Reagent              
-
    10xPyrobest bufferII        10x                 5      10
+
|Concentration/Activity        
-
    Pyrobest                                     0.3     0.5
+
|align="right"|50ul     
-
    dNTPmix               10mM each             1     2
+
|align="right"|100ul
-
    Primer 1               10uM                     1     2
+
|-
-
    Primer 2               10um                     1     2
+
|10xPyrobest bufferII         
-
    Template DNA           changeable             0.5     1
+
|10x                  
-
    MgCl2               0.2M                     0.5     1
+
|5       
-
    ddH2O                 ---                   40.5     81
+
|10
-
 
+
|-
 +
|Pyrobest
 +
|---                                      
 +
|0.3      
 +
|0.5
 +
|-
 +
|dNTPmix                
 +
|10mM each              
 +
|1      
 +
|2
 +
|-
 +
|Primer 1                
 +
|10uM                      
 +
|1      
 +
|2
 +
|-
 +
|Primer 2                
 +
|10um                      
 +
|1      
 +
|2
 +
|-
 +
|Template DNA            
 +
|changeable              
 +
|0.5      
 +
|1
 +
|-
 +
|MgCl2                
 +
|0.2M                      
 +
|0.5      
 +
|1
 +
|-
 +
|ddH2O                  
 +
|---                    
 +
|40.5      
 +
|81
 +
|}
   Progress     Program I                 Program 2
   Progress     Program I                 Program 2

Revision as of 17:48, 28 October 2008

HOME Team Project Parts Modelling Notebook Doodle Board

Basic Wet-lab protocols

PCR Fusion PCR Restriction cut Ligation Transformation

PCR

PCR by Pyrobest DNA polymerase
Reagent Concentration/Activity 50ul 100ul
10xPyrobest bufferII 10x 5 10
Pyrobest
0.3 0.5
dNTPmix 10mM each 1 2
Primer 1 10uM 1 2
Primer 2 10um 1 2
Template DNA changeable 0.5 1
MgCl2 0.2M 0.5 1
ddH2O
40.5 81 
 Progress	     Program I	                 Program 2
   Predenaturing    95℃         2-5 min	         95℃   2-5 min
   Denaturing	     95℃        10-20sec	         95℃   10-20sec
   Annealing	     (Tm-5) ℃   2-5 sec	         68℃   10-15sec/1kb
   Extension	     72℃        10-15sec/1kb

25-30cycle 25-30cycle 

   Last extension	  1-2min or skipped	       1-2min or skipped

Fusion PCR (1) System The basic system is similar to common PCR. There are some notes to raise the fusion efficiency. a. Complementary region length: 15-20bp b. Raise the annealing temperature in the fusion step. (2) Program:

• Program: • 95’: 5min • 95'  : 30-50sec • Tm(fu)+ (-2)~5: 40-80sec • 72' : the longer/1kb/min 10-15 cycles • 72' 5min • Add amplification Primers • 95' 2-5min • Go on under common program for 25-30 cycles


Restriction cut 

           Reagent	        Concentration/Activity	 Volume(50ul system)
           Restriction cut buffer	  10x	           5ul
           Enzyme 1	                --	           1ul
           Enzyme 2	                --	           1ul
      Add DNA and distilled water to 50ul.
 Incubate at 37℃, 1.5 hrs or longer
 (Enzymes from Takara Co., Ltd or NEB)


Ligation 

              Reagent	    Volume(10ul system)
              Solution I	5ul
              DNA fragment	3.5ul(changeable)
              Vector	        1.5ul(changeable)
   Incubate at 16-18℃,1hr or longer
   (Ligation kit from Takara.,Ltd)

Notes: Advanced protocol for parts extraction




  • Click on any day below to see what wet-lab procedures were conducted.
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