Team:Ionis Paris/Protocol 7
Prepare the following reaction in a 0,2 mL PCR tube : Gently mix the reaction and spin down microcentrifuge. 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 10 µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
Protocol 7 : PCR and PCR purification
Aim: DNA fragment amplification and purification
PCR
NB : All components must be vortexed before use
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
58°C 60 s
68°C 1 min per kB to amplify
- Final extension : 68°C 5 min
- Hold : 4°C PCR purification
Place a QIAquick column in a provided 2 mL 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 0.75 mL 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 2 mL 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 30 µL 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.
