Team:Tsinghua/Notebook

From 2008.igem.org

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'''1. PCR'''
'''1. PCR'''
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''1.1 PCR System''
 
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|align="center"|Reagent              
|align="center"|Reagent              
|align="center"|Concentration/Activity        
|align="center"|Concentration/Activity        
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|align="center"|50uL System    
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|align="center"|Volume (50uL System)
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|align="center"|100uL System
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|align="center"|Volume (100uL System)
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|align="center"|10xPyrobest buffer II         
|align="center"|10xPyrobest buffer II         
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''1.2 PCR Program''
 
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'''2. Fusion PCR'''
'''2. Fusion PCR'''
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''2.1 System''
 
The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.
The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.
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''2.2 Program''
 
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'''3. Restriction Digestion'''
'''3. Restriction Digestion'''
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|Reagent          
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|Concentration/Activity  
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|align="center"|Reagent          
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|align="right"|Volume(50ul system)
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|align="center"|Concentration/Activity  
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|align="center"|Volume(50uL system)
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|Restriction cut buffer    
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|align="center"|DNA    
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|10x            
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|align="right"|5ul
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|align="center"|<1ug
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|Enzyme 1
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|align="center"|Restriction Enzyme buffer  
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|align="center"|10x          
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|align="center"|5uL
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|align="center"|Enzyme 1
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|align="right"|1ul
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|align="center"|Enzyme 2
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|align="right"|1ul
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|align="center"|1uL
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|align="center"|ddH2O 
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|align="center"|to 50uL
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Add DNA and distilled water to 50ul.Incubate at 37℃, 1.5 hrs or longer(Enzymes from Takara Co., Ltd or NEB).
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Incubate at 37℃, 1.5 hrs or longer(Enzymes from Takara Co. Ltd or NEB).

Revision as of 11:42, 29 October 2008

HOME Team Project 1 Project 2 Parts Modelling Notebook Doodle Board

Basic Wet-lab protocols

PCR Fusion PCR Restriction cut Ligation Transformation


1. PCR


PCR System (By Pyrobest DNA polymerase)
Reagent Concentration/Activity Volume (50uL System) Volume (100uL System)
10xPyrobest buffer II 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


PCR Program (By Pyrobest DNA polymerase)
Step Condition Time
1 95℃ 5min
2 95℃ 30sec
3 [Tm(fu)-4]℃ 30sec
4 72℃ DNA length/kb/min
5 RETURN TO STEP 2 30-35 cycles
6 72℃ 10min
7 4℃ HOLD



2. Fusion PCR


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.


Fusion PCR Program
Step Condition Time
1 95℃ 5min
2 95℃ 30-50sec
3 {Tm(fu)+[(-2)~5]}℃ 40-80sec
4 72℃ DNA length/kb/min
5 RETURN TO STEP 2 10-15 cycles
6 72℃ 5min
7 Add amplification Primers
8 95℃ 2-5min
9 95℃ 30sec
10 [Tm(fu)-4]℃ 30sec
11 72℃ DNA length/kb/min
12 RETURN TO STEP 2 25-30 cycles
13 72℃ 10min
14 4℃ HOLD


3. Restriction Digestion

Restriction Digestion System
Reagent Concentration/Activity Volume(50uL system)
DNA <1ug
Restriction Enzyme buffer 10x 5uL
Enzyme 1 1uL
Enzyme 2 1uL
ddH2O to 50uL

Incubate at 37℃, 1.5 hrs or longer(Enzymes from Takara Co. Ltd or NEB).


4. 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

BioBrickTM Parts Making Protocol 1. get desired sequences through NCBI or other sources and check for restriction sites For no (Xba1 or Spe1) Goto STEP2 Else Goto STEP9 2. Design primers with half-prefix (Xba1) and half-suffix (Spe1) 3. PCR from according genome/plasmid 4. Purify PCR product using Gel Extraction Kit (Transgen) 5. Digest with Xba1+Spe1 6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 and treated with CIAP 7. Transform to TOP10 cells 8. Identify clones with colony PCR Goto STEP20 9. Design primers with full-prefix and full-suffix 10. PCR from according genome/plasmid 11. Purify PCR product using Gel Extraction Kit (Transgen) For EcoR1 Goto STEP12 Else Goto STEP14 12. Digest with Xba1+Pst1 13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 and treated with CIAP Goto STEP16 14. Digest with EcoR1+Spe1 15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 and treated with CIAP 16. Transform to TOP10 cells 17. Identify clones with colony PCR 18. Extract plasmid and site-directed mutate by fusion PCR 19. Transform to TOP10 cells 20. Extract plasmid and send sequencing End^^67



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