Team:HKUST/Protocols/PCR

PCR

Purpose: To amplify a specific piece of DNA out from the whole.

Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent. Taq gives sticky ends after PCR, while Vent gives blunt ends.

Procedure:

1. Add 10 μL water to make it a 20 μL reaction.
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase
3. Vortex and then spin down.
4. Put it into the PCR machine and set the program.
  Program
    (1) Initial denaturation 95 °C 4 mins
    (2) Run 25-30 cycles of:
    Denaturation   95 °C 30 secs
    Annealing     30 secs
      Temperature is depended on melting temperature of primer.
      Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent
    Extension 72 °C 30 secs per 500bp PCR product length
(3) Final extension   72 °C 3~5 mins

Tips:

The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing