Make up to mL with water; Acrylamide from Biorad. Adjust pH to 8. Make up to 1 Liter with water 1. SDS from Biorad. Adjust pH to about 6. Make up to 1 Liter with water 0. Electrophoresis involves the application of potentially dangerous voltages to the gel and gel reservoirs.
At no time should any part of the electrophoresis set-up be touched following application of current to the apparatus. Step 3: Sealing the Gel Box the following instructions are for a vertical gel box see "Electrophoresis of Dyes" representing an example of a horizontal run.
Stacking Gel Solution A-- 1. The following steps involve the preparation of the gel for data analysis. It is recommended that gloves be worn at all times. Staining Solution 1. Students are to plot the relative mobility as compared to bromophenol blue RBPB for each of the marked proteins.
The data are generated from the start of the running gel. Students will plot the RBPB against the log of the molecular weight for each protein marker. The molecular weight of an unknown protein bands can then be determined. Once determined the students will calculate the percent error for known values of the protein bands.
After class discussion of results, the concept of using a linear gradient will be either dynamically generated or funneled by the teacher.
Students will be given two 10 cc syringes, various tubing, clamps etc. The students will also be given an article take from Electrophoresis in Practice by Reiner Westermeier summarizing the effect of using a linear gradient.
After testing their system, groups will poster their results. A group presentation will follow where each group will explain their system and results. Each member of the group will be assigned a particular section of the presentation: 1 Discussion of design; 2 Presentation of data; 3 Comparison of data to prior nonlinear gradients; and 4 Sources of error, plus the highlighting the areas of improvement in the design. The electrophoretic technique used here employs polyacrylamide gels.
This technique exploits differences in molecular size and charge for the purposes of separation. The gel components are not charged and can be varied in a known manner to produce gels of various specific pore sizes.
An effective pore radius of 0. Thus, it is possible to redissolve the portion of the gel containing a macromolecule of interest.
Gelation takes place more slowly at lower pH values because the free form of the base is required to catalyze the reaction. The polymerization rate is highly temperature dependent, hence the temperature must be kept constant.
Electrophoresis of Proteins Since proteins are amphoteric, the pH of the electrophoresis system must be chosen bearing in mind the isoelectric points of the proteins which are to be separated.
The electrophoresis system in this experiment employs a detergent, sodium dodecyl sulfate SDS. The SDS system involves concentrating protein samples into very thin layers using a discontinuous voltage gradient and then electrophoresed onto a column of polyacrylamide gel. The concentration step enhances the final resolution obtained. Principles of Electrophoresis The protein concentrates because the solvent is made discontinuous. Chloride and glycine solutions are electrophoretically in series.
The movement of ions the current must be the same throughout the system. When a voltage is applied the glycine and chloride will move toward the positive electrode. This concentrating step is called "stacking" and occurs in the upper gel. A tracking dye is added to the protein or the upper buffer before electrophoresis. It has a mobility which is dependent on the pH in this region and is intermediate between chlorine and glycine.
It is customary to indicate the relative mobility of substances on the gel as a ratio of the distance traveled by the substance to that traveled by the tracking dye.
Agarose electrophoresis is the standard method for separation, identification and purification of DNA and RNA fragments. Horizontal gels are used for these nucleic acid separations: the agarose gel lies directly in the buffer.
This prevents the gel from drying out. The gels are stained with ethidium bromide and the bands are visible under UV light. Using proteins called restriction enzymes, genes can be cut at specific DNA sequences. More than 75 different kinds of restriction enzymes are known, and each one "recognizes" and cuts DNA at a particular sequence. The accuracy of these enzymes is amazing. They will not cut any sequence other than the one they recognize, even if five out of six base pairs are identical to their recognition site.
Restriction enzymes make it possible to cut DNA into fragments that can be isolated, separated, and analyzed. Cooperative learning increases achievement, stimulates cognitive development, promotes active learning, increases self-esteem, and enhances positive attitudes toward school. Increasingly, business and industry require that people work together in production teams or in problem-solving teams.
Therefore, learning to work effectively in a group is important; cooperative problem-solving groups in the chemistry lab help students build and hone their skills. Students will work in lab groups of two.
Once completed they will coordinate their presentation of data, as described in the conclusions, with another team. These may be purchased to save preparation time! EDTA 0. Slowly add EDTA while stirring with a stir bar at low heat.
Adjust the pH to 8 and autoclave. Ethidium Bromide soln. Students are to assign sizes to the DNA bands that they see, using the restriction map of phage lambda DNA as a guide. Students are to record their results in their journals by graphing the samples based on the log of the marker size vs. From this data, students are to determine marker sizes for the bands produced by BamH1. Each group of four students will be subdivided into two lab stations two groups of two. The small groups of two will run the gel using the same set of conditions in order to compare their results when completed.
Each large group of four will be given a sample that contains one similar EcoR1 or HindIII and one dissimilar restriction enzyme. Groups will post their results during a group presentation explaining similarities and differences in the data generated using the poster technique. Each member of the group will be assigned a particular section of the presentation: 1 Summary of lab experience, 2 Presentation of data, 3 Comparison of data, and 4 Sources of error with practical application.
It is a little more costly but can save time! The bands of the ladder each contain from 1 to 12 repeats of a 1,bp DNA fragment.
In addition to these 12 bands, the ladder contains vector DNA fragments that range from 75 to bp. The ladder consists of an equimolar mixture of six blunt fragments from to bp. All gel boxes consist of two wells for buffer solution, an elevated area to place gel plates and two electrodes. Some gel boxes have the gel plate or tray immersed in the buffer solution, but we did not find it satisfactory. Absorbent white paper strips can be placed with one end in the buffer well and the other draped over the edge of the gel plate so that a complete connection is made.
Two small plastic containers mL each are placed on opposite ends to serve as buffer wells. Any small plastic pieces can be placed between these two wells as a support for the glass plate with the gel. The author used an overturned plastic basket. It is not necessary to place all these smaller components in a larger container, but it leads to stability and safety.
This author has tried graphite pencils as electrodes and found them satisfactory for a short term. If you want to repeat this experiment for many years, obtain stainless steel strips or rods from a hardware store to use for the cathode. Platinum wire is the best choice for anode, but is not needed for the dye separation If you use platinum wire, 0.
The electrodes chosen can be taped to opposite sides of the larger container. If you have platinum electrodes from a Hoffman apparatus, they should also suffice. Any method to make electrodes secure should be chosen. Finally, you need absorbent paper to function as a salt bridge between the buffer trays and the gel. Double the paper towels for greater ion flow.
Drape the towel over opposite ends of the gel plate. Generally, the pKa value acid dissociation constant should be close to the required pH. It is preferable to us buffers that provide a low charge magnitude so as not to conduct too much current. Pete Collins has been writing since , primarily on data analysis and environmental science topics. How Electrophoresis Works. The Purpose of Electrophoresis.
Steps of DNA Transcription. List of the Applications of Electrophoresis. The Types of Electrophoresis. How to Prepare Buffer Solutions. What Causes Smearing in Electrophoresis? This pH range has also proven efficacious for most forms of DNA electrophoresis. Thus, buffers with a pKa in the range of are best suited for most electrophoretic applications. This type of buffer yields high ionic strength without providing high buffering capacity.
At relatively low concentrations, the gel conducts too much current. Furthermore, with ions moving quickly through the gel, the buffer may become depleted. One of the reasons Tris-borate is a popular buffer for electrophoresis is that both Tris base and borate are uncharged part of the time at the desired pH, which reduces their electrophoretic mobility.
Reduced mobility of the buffer ions allows for high concentrations of the buffer solution to be employed with the consequent benefits to buffering capacity and sample stability without producing unacceptably high conductivity. Having a low charge to mass ratio, Tris moves much more slowly than small ions such as chloride or phosphate. Buffer ions are not sieved by the matrix, so their migration rates are determined solely by their charge to mass ratio.
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