Team:Tsinghua/Notebook

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!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
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'''Basic Wet-lab protocols'''
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PCR
 +
Fusion PCR
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Restriction cut
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Ligation
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Transformation
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PCR
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PCR by Pyrobest DNA polymerase
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Reagent             Concentration/Activity       50ul    100ul
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    10xPyrobest bufferII        10x                 5      10
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    Pyrobest                                     0.3     0.5
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    dNTPmix               10mM each             1     2
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    Primer 1               10uM                     1     2
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    Primer 2               10um                     1     2
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    Template DNA           changeable             0.5     1
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    MgCl2               0.2M                     0.5     1
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    ddH2O                 ---                   40.5     81
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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
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Fusion PCR
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(1) System
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The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.
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a. Complementary region length: 15-20bp
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b. Raise the annealing temperature in the fusion step.
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(2) Program:
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• Program:
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• 95’: 5min
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•     95'  : 30-50sec
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•     Tm(fu)+ (-2)~5: 40-80sec
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•     72' : the longer/1kb/min
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10-15 cycles
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• 72'  5min
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•         Add amplification Primers
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• 95'  2-5min
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•         Go on under common program for 25-30 cycles
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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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            Enzyme 1                 --           1ul
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            Enzyme 2                 --           1ul
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      Add DNA and distilled water to 50ul.
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  Incubate at 37℃, 1.5 hrs or longer
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  (Enzymes from Takara Co., Ltd or NEB)
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Ligation
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              Reagent     Volume(10ul system)
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              Solution I 5ul
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              DNA fragment 3.5ul(changeable)
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              Vector         1.5ul(changeable)
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    Incubate at 16-18℃,1hr or longer
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    (Ligation kit from Takara.,Ltd)
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Notes:
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Advanced protocol for parts extraction
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*Click on any day below to see what wet-lab procedures were conducted.
*Click on any day below to see what wet-lab procedures were conducted.

Revision as of 16:52, 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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