Lab 4_Gel Electrophoresis Manual.pdf

EXERCISE 7: AGAROSE GEL ELECTROPHORESIS Gel electrophoresis is widely used as a means to separate mixtures of nucleic acids and proteins. This laboratory method is usually used for analytical work, e.g. for estimating the size and/or amount of proteins or nucleic acids. Agarose is generally used fo...

EXERCISE 7: AGAROSE GEL ELECTROPHORESIS Gel electrophoresis is widely used as a means to separate mixtures of nucleic acids and proteins. This laboratory method is usually used for analytical work, e.g. for estimating the size and/or amount of proteins or nucleic acids. Agarose is generally used for larger nucleic acids, while polyacrylamide gels can be used as a matrix for separating smaller oligonucleotides. A variety of stains are available to detect nucleic acids in gel under UV light. A common stain is ethidium bromide, a fluorescent molecule which binds specifically to nucleic acids. Newer fluorescent stains are now available such as Sybr Gold, Syber Green I, Sybr Green II and Syber safe; these stains are now used in place of ethidium bromide for their higher sensitivity and less mutagenicity. Molecular size standards are also typically used to estimate the sizes of DNA molecules in a sample. An example of a frequently used size standard is lambda phage DNA digested with HindIII restriction enzyme. LEARNING OBJECTIVE: To learn how to conduct and interpret/analyze the results of an agarose gel electrophoresis experiment. MATERIALS: • • • • • • • • • 0.8% agarose gel Ethidium bromide solution Bromphenol blue indicator Electrophoresis set –up Micropipettes (P2 and P20) and pipette tips Ice and ice bucket Parafilm Microwave Kimwipes METHODS: A. Preparation of the Electrophoresis Buffer & Casting a gel. 1. Prepare the electrophoresis buffer from the concentrated stock. Prepare the agarose gel; 0.8% gel (0.32 g agarose in 40 ml electrophoresis buffer) for analyzing restriction enzyme digests; prepare 1.2% for analyzing PCR products. 2. Microwave this suspension until a uniform solution is formed; Allow this solution to cool for about 20 mins. If the agarose has not completely dissolved, return the flask to the microwave for further heating 36 Caution: The solution is very hot; do not touch it with bare hands or you may get burned. Use hot holders to handle the solution. 3. After cooling the agarose gels, add 0.2 µl of ethidium bromide (EtBr) Note: EtBr is a mutagen and suspected carcinogen; always wear gloves when handling it!! 4. Pour the cooled solution into the electrophoresis tray and allow the gel to polymerize (approximately 30 mins at room temperature). Place the comb to create sample wells. 5. Once the gel is polymerized, remove the comb carefully. 6. Add electrophoresis buffer to the chambers until the buffer level is just above the gel and has filled the wells. B. Loading samples and running the gel 1. Load the gel by drawing the sample in the sample well with the use of a micropipette. A bromphenol blue indicator must be included in each sample, see notes on the next page for loading of samples (bullet no 3) (Note: A previous exercise on practice in loading the gel should have been performed by the students). Obtain the volume of the sample and mix it with the volume of the dye. Mixing can be done in a piece of parafilm. Gently pipette up and down to mix the dye with the sample. Add each of the sample into a different well. Write down the sequence so you can interpret the results. There should be one lane on the gel dedicated to the ladder. *Mixing of sample and the dye can be done in a piece of parafilm; gently pipette up and down to mux the dye with the sample. Then add or load each of the sample into the difference sampling wells. Be sure to write down the sequence of the sample so you could interpret the results. There should be one well dedicated to the DNA ladder. 2. Run the gel (180 V for 7 cm gel should be fine). 3. Stop the gel when the bromphenol blue dye front is about 2 cm from the end of the gel. C. Staining and visualization 1. Remove the gel by carefully supporting the gel to prevent damage to the gel. Place the gel in a plastic tray. 2. Stain the gel with Sybr Safe: add 60 ml electrophoresis buffer to the tray and add 6 microliter of 10000X Syber Safe; incubate in the dark with gentle agitation for 20 mins 37 3. Convey the gel to the UV illuminator and evaluate the gel using 254 nm and document your result. Notes: • Prepare the 1-kb DNA ladder as follows for gel electrophoresis: 4 µl of 6X dye + 18 µl of H20 buffer + 2 µl of 1-kb ladder stock = 24 µl (total volume). This will be enough for two lanes (if running a gel with only one comb, cut the volumes in half). • Prepare each PCR as follows for gel electrophoresis: 2 µl of 6X dye + 6 µl of H20 buffer + 4 µl of PCR sample = 12 µl (total volume). • Load 12 µl of the 1-kb ladder into a single lane of the top set of wells, and load the remaining 12 µl into a single lane of the bottom set of wells. Then load the 16 PCR samples (12 µl each) into the remaining wells. Subject the samples to electrophoresis for approximately 30 minutes at 95 mA. • When electrophoresis is complete, turn off the power supply and then remove the gel from the electrophoresis apparatus. Be sure to wear gloves when manipulating the gel. View the DNA bands using UV gel-imaging system (a transilluminator). Please consult the teaching assistant (TA) or instructor for the instructions on how to use the gel-imaging system. Take a picture of the illuminated gel for your laboratory notebook. Save the gel image as a JPEG file (.jpg extension) for upload to the CURL Dispose of your gel, your gloves, and any other products contaminated with EtBr in the appropriate waste container. • Examine your gel picture. Your DNA bands corresponding to the 16S rRNA amplicon should align with the 1.5-kb ladder. Make sure you glue or tape the picture to a page in your lab notebook, and immediately label each lane with the identity of the sample loaded as well as the size of each DNA fragment. • When you read gels, the orientation of the gel should be such that the wells in which samples were loaded are placed at the top and the order can be read from left to right. The larger fragments should be at the top, and the smaller fragments should be near the bottom. You should label the size of each band in the DNA ladder, since this can vary depending on the manufacturer. Your Lab Instructor will provide this information. You should also label what sample was loaded in each lane and note the size of each fragment in each lane; this can be deduced by comparing the relative migration of bands to those in the DNA ladder. 38 REFERENCE: Thompson D.A. 2011. Cell and Molecular Biology Lab Manual edited by SL Thompson 39