6.4: Part II- Casting Agarose Gel
- Page ID
- 138998
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Agarose is a complex carbohydrate found in seaweed. If agarose is dissolved in a boiling liquid and then cooled, the solution converts into a solid gel matrix. The agarose solution will be poured into a casting tray to form the desired gel shape. A gel comb has teeth that is used to form the “wells” or holes for loading the samples. You will be prepare and cast a 1% agarose gel with electrophoresis buffer.
Materials
- Agarose powder
- Weigh boat
- Spatula
- Masking tape
- 100 Graduated cylinder
- 250 mL Erlenmeyer flask
- Electrophoresis Gel Casting tray
- Gel comb
- Deionized or distilled water
- 1X Electrophoresis buffer
- Heat-resistant silicone mitts or tongs
- Electronic or analytical balance
- Microwave
Method
If you have concentrated electrophoresis buffer stock, you must dilute the stock to 1X working concentration before preparing agarose solutions or running gel electrophoresis.
- For 20X stock, combine 25 mL 20X stock with 475 mL deionized water to make 500 mL 1X buffer.
- For 50X stock, combine 10 mL 50X stock with 490 mL deionized water to make 500 mL 1X buffer.
- Using the graduated cylinder, measure 100 mL of the 1X electrophoresis buffer.
- Using an electronic scale, measure 1.0 g of agarose powder.
- Pour some of the measured buffer into an 250 mL Erlenmeyer flask. Pour in the measured agarose powder. Pour some of the measured buffer into the agarose weigh boat, and pour into flask. Repeat until all agarose has been transferred to flask. Pour rest of buffer into flask.
- Cover the opening of the flask with plastic wrap. Use a pipette tip to poke a small hole in the plastic wrap.
- Place the covered flask in a microwave and heat on high. When you see bubbles form in the solution, pause the microwave, use oven mitts to gently swirl the flask a few times.
- Continue microwaving the flask until the liquid starts to bubble again. Using oven mitts, hold the flask to the light and swirl the solution. Look carefully to check that there are no specks or swirls of agarose suspended in the liquid. If liquid is clear, then the agarose is dissolved. Wait five minutes for the agarose to cool.
Instructor will announce if you have a casting stand system and do not need to tape each tray.
- Prepare the acrylic electrophoresis gel trays by taping the two open ends of each tray. Be sure to press tape firmly along the entire edge of the tray with your fingernail. If using masking tape, you can see a difference in the tape translucence.
- Place a comb in each tray before adding the agarose solution.
- When the agarose solution has cooled to the point that you can safely touch the bottom of the flask (~60°C; about five minutes), pour agarose solution into each casting tray, so that the solution covers about 2 mm of each comb.
Each Mini One gel requires 12.5 mL agarose solution, each casting tray holds two gels = 25 mL total.
- Once the gels solidify (which will take around 30 minutes), pull the comb out of each gel. Pull it straight out without wiggling it back and forth; this will minimize damage to the front wall of the well.