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Procedure
I. Set Up Restriction Digest 1. Use permanent marker to label four 1.5-ml tubes, in which restriction will be performed. E EcoRI, H HindIII, -no enzyme and ? unknown enzyme. 2. Use matrix below as a checklist while adding reagents to each reaction tube. Read down each column, adding the same reagent to all appropriate tubes; use a fresh tip for each reagent. Tube DNA Buffer EcoRI HinduIII H2O unknown E 4 ul 5 ul 1 ul ---------- --------------H 4 ul 5 ul ------1 ul --------------- 4 ul 5 ul ---------------- 1 ul ----------? 4 ul 5 ul ---------------- ----1ul 3. Close tube tops. Pool and mix reagents by pulsing in a microfuge. Make sure tubes are placed in a balanced configuration in the rotor. 4. Place reaction tubes in 37oC water bath, and incubate for a minimum 20 minutes. Reactions can be incubated for a longer period of time. II. Cast 1% Agarose Gel 1. Seal ends of gel-casting tray with tape, and insert well-forming comb in the notches over the black band. Place gel-casting tray out of the way on your lab bench so that agarose poured in the next step can set undisturbed. 2. Carefully pour enough agarose solution into casting tray to fill to depth of about 5mm. Gel solution should cover only about 1/3 the height of comb teeth. Use a pipet tip to move large bubbles or solid debris to sides or end of the tray, while the gel is still liquid. 3. Gel will become cloudy as it solidifies (about 10 minutes). Be careful not to move or jar casting tray while agarose is solidifying. Touch corner of agarose away from comb to test whether gel has solidified. 4. When agarose has set, unsea. Ends of casting tray. Place tray between pegs in the gel box. The comb should be at the negative end (black). 5. Fill box with 1X Tris-Borate-EDTA (TBE) buffer, until it just covers the entire surface of the gel. Too much buffer will channel the current over the top rather than through the gel, increasing time required to separate DNA. 6. Gently remove comb, taking care not to rip wells. Do not wiggle the comb as you pull it out. 7. Make certain tht sample wells left by the comb are completely submerged. If dimples are noticed around wells, slowly add buffer until they disappear.
III. Load Gel and Electrophorese 1. Add 1 ul of loading dye to each reactiontube. Mix by pulsing in a microfuge. Make sure tubes are placed in a balanced configuration in rotor. 2. Use a micropipet to load 10 ul of each reaction tube into separate well. Use a fresh for each reaction. a) Steady pipet over well using two hands. b) Dip pipet tip through surface of buffer, center it over the well, and gentey depress pipet plunger to slowly expel sample. Sucrose in the loading dye increases the density of the DNA mix so it stinks to the bottom of the well when loaded. Be careful not to punch the tip of the pipet through the bottom of the gel. 3. Close top of electrophoresis chamber, and connect electrical leads to a power supply, anode to anode (red-red) and cathode to cathode (black-black). Make sure both electrodes are connected to the same channel of power supply. 4. Turn power supply on, and set to 130 volts. 5. Electrophorese for about 45 minutes. Shortly after current is applied, loading dye should be seen moving toward the positive side of the gel. 6. Turn off supply, disconnect leads from the inputs, and slide off topof electrophoresis chamber. 7. Label staining tray with your name. Remove casting tray from the gel box, pour off excess buffer, and slide get into disposable staining tray. The gel is sippery , so be sure to hold the casting tray carefully. 8. Give your gel to the instructor for staining.
2. What is the function of restriction buffer? Restriction enzyme buffers have the appropriate salts and sometimes include essential co-factors that allow the enzyme to function. If you weren't to use the appropriate buffer, chances are the enzyme didn't work, the DNA didn't cut appropriately and your conclusions about the DNA you use will be wrong. 3. What are the two functions of loading dye? First, loading dye is meant to help weigh down the DNA, so that it can sink into the bottom of the wells and not float in the buffer solution. It forces the dye to move more quickly than the actual DNA parts so it is an indicator to when to turn off the power on the electrophoresis chamber.The dye also makes the DNA visible to the naked eye, giving it a purplish color, and making it easier to work with. 4. How does ethidium bromide stain DNA? How does this relate to the need to minimize exposure to humans? EthBr intercolates between base pairs in DNA. If the EthBr is not properly handled with the DNA and in the wrong place and you may create a cancerous cell. 5. What is the effect of run time on the observed pattern of restriction fragments? How would the gel concentration affect the observed pattern? The longer the run time is, the more apart the DNA fragments will be which will allow for better observations. However, you dont want it for too long that the DNA falls off. The gel concentration would increase as the run time gets longer. 6. What is the identity of the unknown restriction enzyme? EcoRI
Possible Errors
Double Digest Too many enzymes caused by not changing the tip each time. Puncturing the gell which causes the lose of the sample.
Forgot to add dye, which will cause the DNA to not be seen. Improper measurement.