Team:Tsinghua/Notebook

From 2008.igem.org

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  Progress     Program I                 Program 2
 
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    Predenaturing    95℃        2-5 min         95℃  2-5 min
 
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    Denaturing     95℃        10-20sec         95℃  10-20sec
 
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    Annealing     (Tm-5) ℃  2-5 sec         68℃  10-15sec/1kb
 
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    Extension     72℃        10-15sec/1kb
 
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                  25-30cycle               25-30cycle
 
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    Last extension   1-2min or skipped       1-2min or skipped
 
Fusion PCR
Fusion PCR
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Restriction cut
Restriction cut
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            Reagent         Concentration/Activity Volume(50ul system)
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            Restriction cut buffer   10x           5ul
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|Reagent          
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            Enzyme 1                 --           1ul
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|Concentration/Activity  
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            Enzyme 2                 --           1ul
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|align="right"|Volume(50ul system)
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      Add DNA and distilled water to 50ul.
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|Restriction cut buffer    
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|10x            
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|align="right"|5ul
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|Enzyme 1
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|align="right"|1ul
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|Enzyme 2
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Add DNA and distilled water to 50ul.
   Incubate at 37℃, 1.5 hrs or longer
   Incubate at 37℃, 1.5 hrs or longer
   (Enzymes from Takara Co., Ltd or NEB)
   (Enzymes from Takara Co., Ltd or NEB)

Revision as of 17:55, 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(Deletable) 0.2M 0.5 1
ddH2O 40.5 81

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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