Preparation of a DNA Sequencing Gel
互联网
实验步骤
1) Locate two LiCor plates (one with a notch and one without) and clean them as follows:
a. Scrape off the polyacrylamide with a razor blade
b. Scrub both sides of the plate with detergent and water using crub brush and wet paper towel.
d. Set the plates on the drying rack to drain.
e. Dry the plates completely using kimwipes (not paper towels)
1) Make the gel mix in a 50 mL polypropylene tube.
3.0 mL of Long Ranger Acrylamide Mix (50% Acrylamide)
Mix until the urea is completely dissolved.
2) Filter the gel mix using a 0.22 um pore filter unit and a 30 mL syringe.
2) Pull the stopper out of the syringe, fill the pipettors with 100 uL APS and 10 uL TEMED.
4) Pour the gel, being careful not to introduce bubbles (bang on the plates while pouring).
4) Make 1000 mL of 0.6x TBE buffer and fill the buffer chambers.
5) Use the syringe with the bent needle to get rid of bubbles clinging to the bottom of the plates.
7) Run the gel for 45 minutes.
5. Denature the sequencing reactions and prepare them for loading.
1) Place the reactions in a heat block at 90℃ for 5 minutes and cool them quickly on ice.
3) Store on ice in dark until ready to load.
6. Loading and running the gel
2) Use a pasteur pipet to flush out the urea which has diffused from the gel into the top well.