Line 74: | Line 74: | ||
<li>Initial denaturation : 95°C 30 s </li> | <li>Initial denaturation : 95°C 30 s </li> | ||
<li>25 cycles of : 95°C 30 s <br/> | <li>25 cycles of : 95°C 30 s <br/> | ||
− | + | Tm* 60 s<br/> | |
68°C 1 min per kB to amplify</li> | 68°C 1 min per kB to amplify</li> | ||
<li>Final extension : 68°C 5 min</li> | <li>Final extension : 68°C 5 min</li> | ||
− | <li> Hold : 4°C</li>< | + | <li> Hold : 4°C</li></p> |
+ | <p> *precise Tm for each primer </p> | ||
<h4 class="blog" style="margin-top:20px"> PCR purification</h4> | <h4 class="blog" style="margin-top:20px"> PCR purification</h4> |
Revision as of 17:03, 16 October 2016
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 :
Tm* 60 s
68°C 1 min per kB to amplify
*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.