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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 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.
