6.6: Part IV- Gel Electrophoresis
- Page ID
- 139000
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Gel electrophoresis is a technique to use electrical current to separate a mixture of molecules such as DNA, RNA, and proteins. The electrophoresis buffer contains ions to conduct electric current. As DNA molecules are negatively charged, they will migrate towards the positive electrode (red). The solidified agarose gel matrix will have pores of various sizes (similar to a sponge), so the size, shape and charge of the molecules can affect the rate of travel through the agarose gel. Smaller molecules move faster than the larger molecules.
DNA can be visualized with various dyes. Scientists typically use ethidium bromide (either inside the agarose gel or as post-stain after the gel run). As ethidium bromide is mutagenic, we will not be using that in our class. Instead, we will use gel green stain, which is compatible with the blue LED transilluminators (eg. MiniOne). The alternative stain is gel red, which works with the uV transilluminators.
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
Setting Up the DNA Samples
- Find your tubes from the restriction digest (Part 1).
- Add 2 uL of Gel green Loading dye into each of the sample tubes. Pipet up and down twice to mix the liquid.
- Place tubes in a balanced configuration in a MicroCentrifuge and spin for five seconds.
Setting Up the Electrophoresis System
- Watch videos and follow instructions for placing the gel tray into the electrophoresis buffer tank.
- Fill the buffer tank with 1X Electrophoresis buffer, ensuring that the entire gel is completely submerged. You want about 1 mm liquid layer above the gel, but not too much buffer as that can build up resistance.
- Check that the gel is oriented with sample wells closest to the negative electrode (black). Check that the power cord can reach easily. Check that the gel box will not need to be moved for 30 minutes.
- Draw and label in your notebook how the samples will be loaded in the gel. Check whether you will be sharing the gel with another group.
- Using a new tip for each sample, load the DNA samples carefully into the gel wells.
- When all the samples have been loaded, close the cover over the electrophoresis box. [Note: Gel green is especially sensitive to light, so do not leave the Mini One light on during the electrophoresis].
- Connect the electrical leads to the power supply. Connect both leads to the same channel, with cathode (–) to cathode (black to black) and anode (+) to anode (red to red). [Note: Mini One system must have orange hood in place to turn on].
- Turn on the power supply and set the voltage to 130–135 V. [Note: Mini One system do not have adjustable voltage].
- After turning on the power on the gel boxes, look for bubbles forming on the negative electrode (to show electric current) and that dyes are moving toward the correct direction. If running the wrong way, wait until dyes are inside the agarose gel, then turn the gel 180o and restart the run..
- Do not allow the loading dye to run off the gel. Be sure to turn off the power switch and unplug the electrodes from the power supply. Do this by grasping the electrode at the plastic plug, NOT the cord.
- Carefully remove the cover from the gel box and pick up the gel tray. Slide the gel onto plastic wrap on top of the appropriate transilluminator. Take a photo.
- Compare the sizes of the DNA ladder to the pUC19 fragments.
- The pPSU1 cut with Pst1 has fragments of 4100, 2000, 1000, 900, 800, 700, and 500.
- The pPSU2 cut with Pst I have sizes of 4100, 1500, 600, 500, 400, 300, 200, 100, 50.
- What sizes are the pUC19 DNA fragments?
Results
Figure 7: Restriction digests after electrophoresis