ANG 111 Summer 2009 Laboratory 0: Basic Laboratory Procedures Objective: To provide a foundation in basic laboratory techniques and safety issues Overview: Students enter this laboratory with a wide range of background information and experience. This session will be devoted to ensuring that everyone is comfortable with performing common procedures used in a molecular genetics laboratory. 1. Safe Laboratory Practices For your protection (and ours), we will review several safety protocols for this laboratory. a) The fire extinguisher is located The safety shower is located The eye wash is located The nearest telephone is located The number to call in an emergency is The fire alarm is located b) In case of skin or eye contact with hazardous materials, the area should be soaked for at least. c) The fume hoods are for storage and use of flammable and toxic chemicals only. They are to be kept clear and immediately cleaned when spills occur. d) Clothing: Open toed shoes (yes, sandals) are NOT permitted in the lab. Wear the appropriate gloves in order to protect yourself from heat and hazardous chemicals. Latex gloves, although vital for preventing contamination of samples from you, will not protect you from hazardous materials. When in the hallway, please make sure at least one hand does NOT contain a glove. e) No food, drink, or smoking is permitted in the laboratory. f) No unsupervised work in the laboratory. g) Clean up after yourself. As you work, keep your area clear of unnecessary chemicals, paper, and glassware. h) Waste disposal: No explosive, flammable, or toxic materials can be tossed in the trash cans or down the drain. We will provide the proper disposal containers. Dispose of glass and sharps in labeled containers as provided. Bacterial culture plates will be autoclaved before disposal. No food related trash is permitted in the laboratory trash cans. In case of an emergency, let one of us know. Do not try to fix things on your own. 1
2. Equipment, Hazardous Materials, and Some Items Deserving Special Attention UV Light phenol/chloroform high voltage hot agarose balancing the centrifuge open flames cross-contamination enzyme longevity Note: We will NOT be using Ethidium Bromide (EtBr) or radioactive isotopes. 2
3. Gel Electrophoresis Gel electrophoresis is a technique that is vital to most molecular genetic work. In this exercise you will prepare an agarose gel. You will then load a DNA marker (that we will use throughout the quarter) into the gel s wells and apply an electrical current in order to separate fragments of different molecular weights. The gels will contain SYBR Safe, a green fluorescent DNA gel stain to allow visualization of the DNA after it has been run. In this procedure, each group will prepare one 50 ml minigel. You will be making an agarose gel of 0.8%, 1.0 % or 2.0% agarose. Molecular biologists use gels ranging from 0.5% to 2% agarose depending on the sizes of the DNA fragments that they want to separate. The percentage is a weight percentage. 1% is defined as one gram in 100 mls. So since you are making a 50 ml gel, you will need to weigh out 0.5 grams of agarose for a 1% gel. The agarose will be dissolved in 1X SB buffer (Sodium Borate). The X in the name of the buffer refers to the strength of the buffer. Or put another way, the concentration of the buffer. For many applications, we make concentrated stock solutions (rather than weighing out minute quantities of chemicals). We designate the concentrations in terms of X for the word times. Thus, a 1000X SYBR Safe solution would be a stock solution that is 1000-times more concentrated than we would use it as. To make a 1X solution, this stock solution would need to be diluted 1000-fold (one part of the concentrated stock plus 999 parts diluent). Recipes for Agarose Gels: % Gel Agarose 1X SB SYBR Safe (1,000X) (weight) (volume) (volume) 0.8 % agarose 0.40 grams 50 mls 50 l 1.0 % agarose 0.50 grams 50 mls 50 l 2.0 % agarose 1.00 grams 50 mls 50 l A) Prepare the gel tray by first adjusting the depth of the gel comb. You want the wells deep enough to hold your sample, but not so deep as to go through the bottom of the gel. They must form wells, not holes. Next place a piece of tape across each end of the gel tray, sealing the tape against the plexiglass of the tray, but leaving enough tape to enable you to remove it once the gel is set. Set the comb in place. B) Mix the correct amount of agarose with the correct SB volume in a 250-300 ml Erlenmeyer flask for the 50 ml gels. Use a bigger flask for a larger gel volume. Place the flask in the microwave and turn the oven on at full power. Watch through the door until the buffer begins to boil. Before the buffer boils over, open the door, and, using the gloves/hot hands provided, gently swirl the mixture. Swirling too fast can cause the agarose to boil explosively, and hot agarose burns badly. Repeat until you do not see any pieces of agarose left in the solution. However, you do not want to boil it too long or else the concentration will be altered (you will have boiled off buffer volume and the gel will be more concentrated). 3
C) Remove the flask from the oven carefully once all the agarose is dissolved. Set the flask on the bench to cool a bit. Mix by gently swirling. Gently because vigorous swirling will result in bubbles that are difficult to remove and interfere with the DNA migration. D) Let the flask cool until it can be held in your bare hand, add the SYBR Safe solution and swirl (10 l SYBR Safe /10 ml gel). Slowly pour the mixture into the gel tray without introducing bubbles, or dislodging the comb. Let the gel stand in the tray until it is fully set (firm to the touch and with an opaque cast to it). E) Carefully remove the comb (pull straight up with one hand while holding the gel in the tray with the other hand) and the end tapes (do not let the gel slip out of the tray while you are removing the tapes). Place the tray into the gel box. Fill the reservoir with 1X SB buffer and completely submerge the gel by 1-2 mm. F) The volume of DNA solution you will load in the gels throughout the quarter depends on the concentration of the DNA. If the DNA is very concentrated, you will usually add some kind of buffer to increase the volume for ease of loading. The marker DNA has been mixed with a loading buffer and dye mixture. The dye mix allows you to track relative DNA migration while the voltage is applied without using the UV light and the loading buffer serves to weigh the DNA sample down in the bottom of the well. For each marker, you should always load 10 l onto the gel unless otherwise instructed. Do not load bubbles in your well and be careful not to blow the sample out the well. G) Once the DNA is loaded, you want to begin applying electrical current quickly, so that your DNA does not diffuse out of the wells into the reservoir. Place the top onto the gel tank (electrophoresis box) and plug in the leads. Connect the red (positive) lead to the electrode at the end furthest from the wells. Plug the leads into the appropriate sockets in the power supply, set the voltage to 200 V and turn the power on. Remember: RUN TO RED. H) Run the gel for about 25 minutes. At the conclusion of the run, turn the power off and remove the top of the electrophoresis unit. Carefully remove the gel tray (the gel is slippery and can leap onto the floor and break into pieces), place the gel without the tray onto a piece of Saran Wrap, and visualize on the ChemiImager. I) The gel with the markers should look like this: bp A B C 23,130 9416 6557 4361 2322 2027 564 Lane A Lambda HindIII Marker Lane B 1 kb ladder (10 kb to 1 kb) Lane C 100 bp ladder (1000 bp to 100 bp) 10 0 4
4. Pipetting Repeatable, accurate dispensing of solutions is an essential laboratory skill for molecular biology work. We will use three micropipetters in this class: 0.5-10 l (white or clear top) using CLEAR tips 10-100 l (yellow top) using YELLOW tips 100/200-1000 l (blue top) using BLUE tips Use the pipetter whose range includes the volume you need to pipet. Check that the volume you have drawn into the tip corresponds to what you need it to be NEVER, EVER, EVER try to pipet a volume that is beyond the range of the pipetter. This will ruin an expensive piece of equipment as well as giving you an incorrect volume. Each group has a set of three pipetters in their lab tray. Each student will pipet, using your group s pipetters, the prescribed volumes of water and weigh them. You will then be able to assess your pipetting technique and the precision and accuracy of the equipment. Question: What is the difference between precision and accuracy? Each student should practice this technique until he/she can pipet accurately. Accurate pipetting is of major importance for getting good results in this course!! 1000 l = 1 ml 1 ml = 1 cc (cubic centimeter) 1 cc of water weighs 1 gram. Weigh three times: 10 l H 2 0 with the 10 l pipetter average 100 l H 2 0 with the 100 l pipetter average 1000 l H 2 0 with the 1000 l pipetter average 5