Lab Tips
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Lab Tips
Plant Pigments and Photosynthesis
Cell Respiration and Fermentation
DNA Extraction
1. DNA from strawberries – strawberries have lots of DNA (octoploid –8 copies of each chromosome), cell walls are partially broken down already in ripened fruit (from cellulases and pectinases)
a. put a fresh ripe strawberry (thawed frozen strawberries will work too) into a ziplock sandwich or freezer bag (thicker bags won’t split)
b. mash the strawberry
c. pour some salt into the baggie (1+ tsp) and a squirt of dishwashing detergent (such as Dawn), precise measurements aren’t that important (may make up a salt/soap soln of 100ml shampoo or 50 ml Dawn to 15g salt in 900ml water)
d. mix well by kneading the bag
e. pour through a funnel containing a coffee filter, cheesecloth, or filter paper into test tube or small glass beaker (easiest way to do this is cut corner of baggie and squeeze out)
f. hold tt or beaker at an angle and slowly pour in ice-cold 95% ethanol (91% isopropyl alcohol/rubbing alcohol may also be used, NOT 70%)
g. the DNA will ppt at the alcohol/filtrate interface
h. let it sit for a few minutes, then spool the white DNA ppt on a glass rod, wood splint, or paper clip with a slow twirling motion
2. Human check cells – best done if they have recently eaten or chewed gum.
a. vigorously swish 10 ml of Gatorade (or use 8% salt solution) around in mouth for 10-15 seconds, then spit it back into a cup and transfer into test tube
b. add 1 ml of a 25% dishwashing detergent (such as Dawn) to the test tube
c. covering the end of the test tube, invert the tube 5 times (or use screw top tubes)
d. pour approximately 5 ml of ice cold 95% ethanol or 91% isopropyl alcohol gently down the side of the test tube (hold tube at 45o angle)
e. you should see DNA at the alcohol/Gatorade interface
3. Bananas (or Apples, Kiwis, almost any fruit)
a. peel and place ripe banana into blender, cover with 15% NaCl and 2 drops of dish soap
b. blend thoroughly
c. pour through several layers of cheesecloth into beaker
d. add a pinch of meat tenderizer
e. pour into test tubes (half full), let settle (15-20 minutes)
f. pour ice cold 95% ethanol in layer above banana mixture
g. at interface DNA should become visible, spool onto glass rod or wooden splint
h. OR blend banana with 1 cup of water, add 3 tsp of this banana mix to 1 tsp shampoo, 2 pinches salt, 4 tsp water and mix, filter, then add ice cold EtOH
4. Principles to emphasize
a. texture/consistency – chromosomal DNA is long strands (why we can spool it around rod)
b. solubility – DNA is soluble in water, insoluble in alcohol
c. pH – use pH indicator on sample top show it is acidic (deoxyribonucleic ACID)
d. steps in isolation
- break open/lyse the cells by blending or squishing the fruit
- dissolve the organelle and cell membranes with detergent
- separate DNA from proteins with salt (+ charged Na+ attracted to – charged DNA so DNA sticks together) also causes proteins to ppt out
- if add meat tenderizer (papain) helps to break down proteins including enzymes that might break apart DNA
- filter to separate DNA from other cellular material
- DNA not soluble in ice cold EtOH so all components stay in solution except DNA that ppt out, the colder the EtOH the less soluble the DNA will be
Diffusion and Osmosis
Diffusion
A. Agar Blocks
1. Phenolphthalein
a. Set-up -Mix 1 packet of unflavored Knox gelatin with 1 cup of water (a 3% agar solution will also work-3g agar/100 ml H2O), then add a few drops of phenolphthalein, and a few drops of NaOH.
b. Continue to add enough NaOH and phenolphthalein (main ingredient of Ex-Lax) so that the gelatin mixture remains bright pink after swirling (1%phenolphthalein and 0.2N NaOH work fine).
c. Pour into container (a plastic freezer dish works well) and refrigerate overnight.
d. Measuring- cut cubes of various sizes (1cm to 3cm squares work well, but can make down to .5cm squares)
e. put cubes into a beaker of white vinegar.
f. Experiments- can do time course on a particular size, can do set time (2-5 minutes) on a variety of sizes, or measure the length of time it takes for the cube to become totally clear.
g. Applying math- cut the cubes in half and measure the distance permeated vs. non-permeated, calculate the ratio of those areas, try different shaped cubes, etc.
h. can also look at same volume vs. different surface area such as a 2 cm on a side cube vs. a 1 x 2 x 4 rectangle
i. Note- Measure the blocks quickly after they have been removed from the vinegar as there will be continued diffusion even after the blocks are taken out.2. Red cabbage
a. quarter a head of purple cabbage, put into a pot of dH2O and bring to a boil. Cool the purple water OR chunks of fresh purple cabbage may be put into a blender with water, pureed, and then strained through cheesecloth
b. substitute this red cabbage pigment extract for the water needed in the Knox gelatin recipe, let harden overnight and cut into cubes
c. When the cubes are placed in white vinegar the color change is from purple to pinkB. Potatoes
1. Alternative- use different sized cubes of white potato and soak them in 1% potassium permanganate. Slice the cubes and measure the distance penetrated vs. non-penetrated
2. soak same size cubes in different potassium permanganate concentrations(.1%, 1%, 5%).Membrane Stress- Beet Lab
1. After cutting and rinsing the beet cores, place them in test tubes to be tested for any of the following potential membrane stressors:
a. Alcohol- propanol, ethanol, methanol may all be used, try different % (0,10,20,30,40 or 0,5,25,50)
b. Salts- NaCl (0,3,6,9,12,15%)
c. Detergents
d. PH
e. Temperature- refrigerator, freezer, room temp, 70, 55, 40 degrees CDiffusion and Osmosis a. Dialysis tubing
1. soak tubing in dH2O at least 30 minutes before use
2. use dental floss to tie tubing or just tie knots in the bag
3. don’t use glucose test tape as it will react with IKI, rather use glucose test sticks for Diabetes or Benedict’s solution for sugar outside of bag (if using this add equal amount Benedict’s and liquid from beaker to test tube, heat in water bath 5-10 minutes or microwave for 2-5 seconds, if positive for sugar the blue will change to rust color)
4. some inexpensive baggies will work in place of dialysis tubing
5. use laundry starch or spray starch or packing peanuts as starch sourceb. Onion
1. Use a purple onion to observe plasmolysis under a microscope, be sure to notice pigmented cells
2. add high salt or sugar soln to slide, observec. Potatoes
1. use a cork borer to make cores of equal size, cut to same length.
2. Use a French fry cutter or a French mandolin to cut the potatoes uniformly
d. Eggs
1. Reference to article in Journal of College Science Teaching, Nov. 1985 called ‘Osmosis and the Marvelous Membrane’: decalcify the eggs in vinegar for 48 hours, then put eggs in unknown solutions (dH2O, .5M sucrose, 1M sucrose, and 2M sucrose), and mass them every 10 or 15 minutes for 1.5 hours. Calculate molarity of the egg- usually about .8M
2. Place the eggs in different types of dye overnight such as methylene blue, Rit dye and food coloring. Each has a different diffusion rate- boil and slice to see the differences
3. Put a raw hen’s egg in white vinegar for 24-48 hours to remove the calcium carbonate shell, wash gently, and you’ll be able to see the translucent membrane. Measure circumference, volume by water displacement, etc. Then place the egg in 250 ml beaker with known volume of dH2O (150 ml), wait 24 hours, and then dry and mass gain in water. Also can mass and put in 100% white Karo syrup, wait overnight and mass again
4. To extend the lab, add food coloring to the distilled water after the egg has been in the syrup for a day or two- this will show that the pigment molecules are smaller than the pores in the egg membraneMisc.
Give each student a peppermint candy and have half the class let the mint dissolve without biting while the other half of the class bites their mint into several smaller pieces and lets those pieces dissolve. Have the students raise their hand when he mint has completely dissolved. The students with the smaller pieces will raise their hands first. Discuss why this happens in relation to surface area/volume
Enzymes
1. Glucose oxidase
a. order glucose oxidase (glucose trinder) from Diagnostic Chemicals Ltd 1-800-325-2436
b. use 1.0 ml enzyme, 1.0 ml of 0.2 mg/ml glucose, and 3.0 ml of dH2O as standard soln when testing temperature
c. when testing substrate concentration, always have 1.0 ml of your total 5 ml volume enzyme, other 4 ml a combination of dH2O and glucose
d. if doing the protein standard curve to determine protein content of enzyme solution you may want to add an extra ml of dH2O to each tube so final volume is 5.0 ml (depends on the spec you are using).2. Lactaid
a. and ONPG
1. make up fresh1.0 mM ONPG
2. crush a lactaid tablet and dissolve in 100 ml dH2O, filter
3. use a 5.0 ml total volume of reaction mixture
4. if doing different temperatures, use 0.5 ml lactaid soln, 3.0 ml of ONPG, and 1.5 ml of dH2O
5. for different substrate concentrations, use 0.5 ml of lactaid soln, and vary volumes of dH2O and 1.0mM ONPG to add up to 4.5 ml (4 ml to 0.1 ml ONPG)
6. read at 400 nm
7. you may also try the experiment with 5 mM ONPGb. Lactaid and glucose testing
1. Lactaid tablets have a coating that contains glucose so will give a positive result in control, to remedy this try to find drops, Lacteeze, may be ordered from digestmilk.com or fitmart.com
2. add drops to milk or lactose and test for presence of glucose (can also use this for respiration lab with yeast, yeast will not metabolize lactose, but if add Lactaid will be able to since lactose will be broken down into useable glucose and galactose)3. Catalase
a. catalase may be purchased from Sigma or Carolina, will keep in freezer
b. baker’s yeast (1 pkg/200ml warm water + tsp sugar)may be used as source of catalase, as can liver (1g/10ml water)
c. use coffee filters or filter paper to cut discs
d. be sure to use fresh hydrogen peroxide
e. can test pH, hydrogen peroxide concentration
4. Amylase
a. spray squares of Bounty paper towels with spray starch, hang to dry
b. fill plastic spray bottle with water and add tincture of iodine so that water has orange color
c. take small bits of samples to be tested and squash on paper towel (positive results given with impatiens flowers, ginger root, banana, bean juice-soak pinto beans over night)
d. let set 5 or so minutes, then spray paper towel with iodine soln
e. if the sample has amylase activity there should be a whit spot on the towel where that sample was squashed
f. alternative – some copy paper and stationary paper have significant starch layer so wouldn’t need to spray with starch to do experiment http://www.science-projects.com/Amylase.htm
Plant Pigments and Photosynthesis
1. Absorption spectrum
a. the peaks for the absorption spectrum of spinach pigments are the same whether extracted by soaking or grinding in acetone, isopropyl alcohol, 95% ethanol, or in boiling ethanol (see figure), you may need to filter
b. compare spectra of pigments isolated from different plants, especially one with varied colors in leaves
c. collect leaves from a particular tree in summer (freeze in plastic bags until needed) and collect leaves from the same tree in the fall, extract pigments and compare spectra of leaves from summer vs. fall
d. cut out the bands of each pigment from chromatogram, soak in a small amount of the solvent, then do absorption spectrum of each individual pigment2. Chromatography
a. make sure the pigments are concentrated if using extracted pigments, put only a thin line of pigment on paper, let dry between each application
b. can use whole leaves, place leaf on paper, roll quarter over leaf several times
c. make sure the chromatography chamber is closed otherwise the solvent will evaporate and leading edge won’t go far so little separation3. DPIP
a. use crisp, fresh spinach
b. place leaves under light for awhile before grinding
c. make sure sucrose soln is chilled
d. don’t over blend, blend 10 seconds, rest 30 seconds, repeat two more times
e. the chloroplasts don’t have to be boiled, heating to 40-45o will inactivate and you should not get the clumping that occurs when they are boiled
f. overhead projectors or slide projectors can be used as a light source
g. use fresh DPIP, it should be very blue4. Fluorescence
a. use fresh leaves (spinach or fresh grass clippings work well), soak one or two handfuls of leaves in 200 ml of 95% ethanol overnight
b. decant off the liquid or filter through cheesecloth into beaker the next morning
c. in a darkened room shine either a blacklight (longwave uv), a bright point source white light such as a halogen flashlight (Mag-light®), or slide projector
d. observe the liquid from a position perpendicular to the light source, it should glow red at the interface5. Starch
a. put geranium or coleus plant in dark for at least a day
b. cut out black construction paper shapes (stars, circles, etc) and attach (paper clips work or glue stick) to Coleus or Geranium leaves
c. place in sunlight for a few days
d. take shapes off leaves and place leaves in boiling ethanol to remove pigments
e. soak leaves in IKI and note light areas (no starch) where shapes have been
f. may also use different colors of cellophane, negatives6. Floating Disks
a. prepare a 0.2% sodium bicarbonate solution (CO2 source), add 1 drop of a dilute liquid soap (breaks surface tension)
b. cut uniform disks with hole punch, cork borer or sturdy straw from leaves, avoiding major veins (use spinach, ivy, plants that have smooth, hairless surface)
c. place disks in 10-20ml of bicarb soln so they won’t dry out
d. pull plunger out of 30 ml syringe (smaller sizes will work) and pour bicarb soln with disks into syringe barrel
e. replace the plunger and push out all air
f. hold finger over syringe-opening and draw back on the plunger to create a vacuum, hold 10 seconds or more
g. release vacuum by taking finger off of syringe opening
h. leaf disks should sink, if some still floating, repeat pulling vacuum
i. place equal # of disks (10 or more) into dishes containing 0.2% bicarb
j. place dishes under conditions testing (different light intensities, different wavelength of light-different cellophanes, etc) with the appropriate controls (dark, water instead of bicarb)
k. time how long it takes disks to float in each condition
l. OR may leave disks in syringes, place in conditions testing, etc
m. Use screw in fluorescent bulbs instead of incandescent bulbs or shop lights so don’t need heat sink
n. Home.earthlink.net/~bioteacher/LeafDisk.htm
Cell Respiration and Fermentation
1. Seeds
a. any type of bean or pea, soaked at least overnight, will work
b. if seeds soaked 2 or 3 days, readings can be taken more often
c. sprouts purchased at the grocery store will work if fresh
d. barley seeds soaked in wet paper towels for a day or two will work well
e. polyester fill works in place of nonabsorbent cotton
f. KOH pellets or soda lime pellets can be used in place of the KOH liquid, place the pellets in the bottom of the vial
g. a drop of food coloring at the end of the pipette makes it easier to see the movement
h. a set-up using a 1ml syringe, stopper, and large test-tube will work well (see notebook for set-up)2. Yeast
a. use 10% glucose
b. 7 g yeast/50 ml tap water, 1 pkg/250 ml/warm water
c. try different substrates – sucrose, saccharin, NutraSweet, fructose, starch, honey, lactose (with and without Lacteeze)
d. try different yeasts- dry active, quick rise
e. see if spices, pH, salts, or temperature have effect
f. see notebook for various set-ups3. Celery and Janus Green (Janus Green B stains mitochondria when in oxidized state, as reduced becomes colorless
a. cut 1cm piece fresh celery with razor blade from outer epidermis and transfer to glass slide with 10% sucrose soln
b. trim away excess celery so have thin slice
c. put on coverslip, look under scope for organelles
d. put a piece of paper towel on one side of slide, add 2-3 drops of Janus Green to other side so stain drawn under coverslip
e. note color of mitochondria and continue to observe as stain bleached out as reduced
Biotechnology
1. Electrophoresis
a. when making agar gels use masking tape to seal the ends of plate
b. pipette a small bead of agarose at the tape/plate interface to seal and allow to harden before pouring the rest of the agar
c. use agar Petri dishes and food dye to practice putting samples into wells
d. use dyes to demonstrate principles of electrophoresis, food dyes, bromphenol blue, and congo red are negatively charged, crystal violet, malachite green, and janus green positively charged
e. add glycerol or glucose to dyes (9 parts dye/1 part glycerol or glucose) so sample will sink into well2. Transformation
a. use BioRad kits for best results
b. temperature shock is critical for success, make sure students keep sample in ice (small sample size means it will quickly warm up if not in ice), carry tubes in ice to 42oC waterbath and quickly place into bath, then remove directly back to ice
c. make sure when adding plasmid they see film (like when blowing bubbles) in loop
d. http://www.glofish.com/
Transpiration
1. Whole plants
Follow same introduction and analysis as outlined in College Board lab manual, but instead use whole plants. Use small plants such as impatiens, begonias, pansies, or any small bedding plant.
a. water plants well that morning before lab
b. remove an entire plant and wrap the entire root ball in a plastic baggie (double bagging is best), tie baggies to stem with string, mark with your group name.
c. pinch of any buds or flowers
d. weigh each successive day for the entire week (it is probably best to start on a Monday) .
e. all groups put their controls in one place, those in front of the fan together, those in bright light together, and the last are misted and covered with another plastic bag and are usually placed with the control. The dark are placed in a drawer.
f. if any leaves fall off, be sure they are put back in the center of the plant to be weighed each day so as not to represent water loss.
g. determine the percent change in mass daily over the week and graph2. Stem and Leaves
a. use a medium sized (250mL) Erlenmeyer flask with a two hole stopper
b. secure a 1.0 or 2.0 mL pipette in one hole using some kind of sealant (aquarium sealant, silicone caulk or warm paraffin work well if you set up the stoppers in advance. Parafilm works well if you want to seal it on the day of the lab)
c. put the stem of a bean plant or woody plant into the second hole of the stopper and seal with parafilm (you may also put a pipette or small piece of glass tubing in the second hole and place the stem in that)
d. fill the flask to the very top with water (adding a drop or two of food coloring makes the pipette easier to read) and, in the sink, slowly but firmly push the stopper in the flask neck. The water will squirt out of the pipette but will retreat slightly when you take the pressure off. Then treat your flasks any way you wish3. Graduated Cylinders
a. place individual plants such as impatiens, including roots which have been rinsed off (so no dirt), into graduated cylinders
b. roots are immersed in water (filled to 100 mL mark with water), stems are then parafilmed in place to create airtight seal.
c. Place in condition testing and record water loss4. Looking at Stomata
a. apply clear fingernail polish to leaf, let dry, then place clear tape over dried area, peel off and look at under scope (lettuce-not iceberg, Transcantia work well)
b. or, place a drop of crazy glue on slide, press the underside of leaf into glue, wait 30-45 seconds, peel off the leaf and you are left with impression of stomata
c. Amaryllis leaves will work well by just peeling off the bottom epidermis with fingernail or forceps and doing a wet mount
d. http://www.biologycorner.com/worksheets/stomata.html
e. http://www.zoo.utoronto.ca/able/volumes/vol-13/3-brewer/3-brewer.htm
f. http://www.classtech2000.com/toucan/modules/stomata/stomata.htm
g. http://www.flinnsci.com/documents/demoPDFs/Biology/BF10226.pdf
Animal Behavior
Pillbugs
1. Experiment
a. test in light vs. dark, test different colors of light by using colored filters or cellophane (there seems to be some aversion to red light). Note – parasites in pillbugs may change the behavior of pillbugs toward light
b. moist vs. dry, rough vs. smooth substrate (try sand, sandpaper, saran wrap, etc)
c. light vs. dark background
d. pH – they seem to like high pH (NaOH)2. Care
a. keep in a plastic container with a lid with plenty of small holes for ventilation
b. cover bottom with moist loose soil and wood chips to hide under c. feed them raw potato, wheat germ, fish food flakes, sliced potatoes, sliced apples, or leaf litter.
d. The biggest problem with pillbugs is dehydration so be sure to mist daily3. Websites
http://insects.ummz.lsa.umich.edu/MES/notes/entonotes3.html
http://web.sau.edu/rlegg/GeneralBiology/Lab%20Isopods%20Objectives.htm4. Read Chapter 4 on Wood lice in Waiting for Aphrodite by Sue Hubbell
Crickets
1. do the same experiments with crickets instead of pillbugs, especially light vs. dark background (check location every 2 minutes for 20 minutes), seem to prefer dark
2. can purchase crickets from most pet or bait storesAnts
1. in the lab keep ants in a clear plastic container with a clear cover with air holes.
2. Different groups test different conditions -light/dark, wet/ dry, or attraction to cotton balls soaked in different solutions (vanilla, sugar water, punch, soap solution) or two food choices ( banana slice versus apple, potato slice/onion slice, Oreo cookie versus vanilla wafer, high carb marshmallow vs. high fat peanut butter).
3. outside do same type of experiment by putting different food choices at same distances from an anthill and count the number of ants that swarm over each type of food (keep size of food same)Termites
Test pheromones by experimenting with the termite trail-following response
1. Experiment
a. natural pheromone chemical of termites is: z,z,e-3,6,8-dodecatriene-1-ol that is released from a gland on the underside of the abdomen. They only need to release about 1 picogram per centimeter for it to effectively mark a trail. The pheromone is airborne and is sensed by smell, not touch. This can be demonstrated by placing tissue between the trail and the insect.
b. certain inks are closely related to this pheromone and elicit the trail-following response, best inks are in papermate and bic pens, Office Depot ball point pens also work (red, green and blue)
c. have students test different inks, different colors, different intensities, different brands, by drawing a line with the pens on paper, put the paper in a container with termites (workers not soldiers) and see if they follow the drawn trail (cut out paper circles and put in Petri dishes)
d. decide on appropriate controls (pencil, felt tip pens, etc)2. Care
a. order termites from Carolina, CT Valley, or try to get free termites from exterminators and then give them back when done
b. keep them in a covered container with rotting wood3. Websites
http://www.ecoed.net/tiee/exps/experiments.shtml
http://www.uky.edu/Agriculture/Entomology/ythfacts/resourc/tcherpln/
termtrails.pdf
http://www.esb.utexas.edu/jcabbott/courses/bio208web/lectures/scimethod/
trail_following_of_termites.htm4. Kit- Carolina Biological 14-3722
Dissolved Oxygen
1. DO kits by Chemetrics (1-800-356-3072) are probably the fastest and easiest way to go with this lab. These may be ordered directly from the company or through Wards or Carolina Biological
2. Hach and LaMotte also makes DO kits