Protocol 3

Protocol 3 : Electrophoresis and gel purification

Aim: Check for correct digestion or cloning procedure or/and isolate on an Agarose gel a specific DNA fragment

Preparation the 1% agarose gel

Add 1g of agarose into 100mL of TAE 1X
Microwave until ebullition
Allow the cooling to 55ºC (you can hold it in your hand) and add 0.1X Gel Red
Pour the liquid gel in its casting tray, equipped with the well combs
Wait until the gel cools down and solidifies

Drop-off

Remove the well combs gently and place the gel+tray in the electrophoresis tank filled with 1X TAE.
Load the wells with of DNA mixed with 1X loading dye.
Make sure the wires and tank are correctly set up. DNA is attracted by the positive terminal.
Set the power source to 80V. Migration time may vary according to the DNA fragment size.
Shut down the power source and remove the gel from the tank.
Visualize the DNA fragments under UV transillumination.

Gel Purification

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Weight the gel slice in a tube. Add 3 volumes Buffer QG to 1 volume gel (100mg gel ~ 100µL). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG.
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel.
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The mixture will turn yellow.
Add 1 gel volume isopropanol to the sample and mix.
Place a QIAquick spin column in a provided 2 mL collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 13000 rpm. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800µL, load and spin again.
If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500µL Buffer QG to the QIAquick column and centrifuge for 1 min at 13000 rpm. Discard flow-through and place the QIAquick column back into the same tube.
To wash, add 750µL Buffer PE to QIAquick column and centrifuge for 1 min at 13000 rpm. Discard flow-through and place the QIAquick column back into the same tube.
Centrifuge the QIAquick column in the provided 2mL collection tube for 1 min at 13000 rpm to remove residual wash buffer.
Place QIAquick column into a clean 1.5mL microcentrifuge tube.
To elute DNA, add 30µL Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min at 13000 rpm.