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. Note: Instructor will announce if you have a casting stand system and do not need to tape each tray.
Prepare the acrylic electrophoresis gel trays for casting. You may need to tape 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. Figure 4. Casting tray with tape Place a comb in each tray before adding the agarose solution. Note: Each Mini One gel requires 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. Part III: Practice Pipetting While you are waiting for the restriction digest to incubate, you can practice loading samples into a practice gel.
Figure 5. Method Watch the video or instructor demonstration. Submerge the practice gel with water or buffer. Do not change tips for this practice. To steady your pipette hand, place your elbow on the table. You may also use your other hand to support and steady your pipette hand. Figure 6. Electrophoresis causes the molecules to separate by size in the porous gel DNA can be visualized with various dyes. 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 a demo or assigned 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. The methylene blue dye will stain skin, clothes, and equipment. Always wear gloves and safety glasses.
Do all staining in a central area near the sink. Restriction enzymes cut at specific sites along the DNA. These sites are determined by the sequence of bases which usually form palindromes. Palindromes are groups of letters that read the same in both the forward and backwards orientation.
The complimentary bases on the opposite strand will be CTTAAG, which is the same as reading the first strand backwards! Many enzymes recognize these types of sequences and will attach to the DNA at this site and then cut the strand between two of the bases. Log In Bookstore Join Renew. It looks like your browser does not have JavaScript enabled.
Please turn on JavaScript and try again. Activity 3: Restriction Enzyme digestion - How does it work? Why is it useful? Page Content. Introduction Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms. Objectives Understand what a DNA restriction enzyme is and how it works. Learn to use a micropipette. Learn to separate DNA on an agarose gel using electrophoresis.
Understand how to use a restriction digestion map to identify a sample DNA. Obtain enough crushed ice and ice containers styrofoam cups for each lab group. Reconstitute the lambda DNA with sterile distilled water to 0. Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed.
Make the 1. Heat in the microwave for 30 seconds at a time, shaking gently each time, until the agarose is completely melted. Alternatively, the solution can be heated on a hot plate, with occasional gentle shaking, until the agarose is melted. Keep warm if the class will use it within a half hour. Otherwise, allow the solution to cool and solidify. Cover and keep in the refrigerator. Pour enough agarose gels for each lab group as follows: Wear gloves Microwave or warm the agarose bottle in a hot waterbath until the gel liquefies.
Be sure to use a microwave designated for science purposes not food. Firmly seal the ends of the gel tray using labeling tape. Place the plastic comb in the slots close to the end of the tray.
Pour approximately ml of agarose into each gel tray. Let cool until solidified approximately 15 minutes. If storing overnight, place trays in a container or ziploc baggie with 0.
The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone.
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