Team:Tsinghua/Notebook

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Basic Wet-lab protocols

PCR Fusion PCR Restriction cut Ligation Transformation

1. 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


2. Fusion PCR

2.1 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.

2.2 Program: 2.2.1 95℃ 5min

2.2.2 95℃ 30-50sec

2.2.3 {Tm(fu)+[(-2)~5]}℃ 40-80sec

2.2.4 72℃ DNA length/kb/min

2.2.5 return to 2.2.2 for 10-15 cycles

2.2.6 72℃ 5min

2.2.7 Add amplification Primers

2.2.8 95℃ 2-5min

2.2.9 continue 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




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