Team:Tsinghua/Notebook

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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.