Team:Ionis Paris/Protocol 7

Protocol 7 : PCR and PCR purification

Aim: DNA fragment amplification and purification

PCR

Prepare the following reaction in a 0,2mL PCR tube :
NB : All components must be vortexed before use

Component 50µL reaction Final concentration
Nuclease-free water to 50µL
10X Standard Taq reaction Buffer 5µL 1X
10mM dNTPs 1µL 200µM
10µM Forward primer 1µL 0.2µM (0.05-1µM)
10µM Reverse primer 1µL 0.2µM (0.05-1µM)
Template DNA <1,000ng
Taq polymerase 0.25µL 1.25 units

Gently mix the reaction and spin down microcentrifuge.
Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C
Set the following parameters for the PCR reaction :

  • Lid température 95°C
  • Initial denaturation : 95°C 30 s
  • 25 cycles of :
    95°C 30 s
    Tm* 60 s
    68°C 1 min per kB to amplify
  • Final extension : 68°C 5 min
  • Hold : 4°C

    • *precise Tm for each primer

    PCR purification

    Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
    Place a QIAquick column in a provided 2mL collection tube.
    To bind DNA, apply the sample to the QIAquick column and centrifuge for 60 s. Discard flow-through and place the QIAquick column back in the same tube.
    To wash, add 0.75mL Buffer PE to the QIAquick column, centrifuge for 60 s. Discard flow-through and place the QIAquick column back in the same tube.
    Centrifuge the QIAquick column once more in the provided 2mL collection tube for 1 min to remove residual wash buffer.
    Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
    To elute DNA, add 30µL Buffer EB (10 mM Tris·Cl, pH 8.5) elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min at 13000 rpm.