BIO 206L Lab Practical Prep

what are the ingredients of the Master Mix?

Taq polymerase, MgCl2, dNTPs, and buffer

what is ball moss

a monocot angiosperm!

Transgenes/Enzyme Station LB + amp + arabinose

- this plate contains bacterial colonies - along with LB and ampicillin, this plate also contains arabinose - the araC/arabinose operon controls the expression of GFP (a gene that comes from a jellyfish), which is what causes these bacteria to fluoresce/glow under UV light - all of these bacteria are transformed/transgenic - they have the GFP gene, because they are glowing - they have the bla gene, because they are ampicillin resistant (surviving on a plate that has ampicillin) - genotype: LB, amp, arabinose - phenotype: fluoresce (b/c arabinose is present in the plate)

setting up an optical dissolved oxygen probe

- this probe measures oxygen content, in mg/L - the probe has a plastic covering on it, take this off - rinse the probe with dH20 and blot dry with a kim wipe - place the probe into the surface of the algal solution (which should be sitting on the stir plate being stirred) - MAKE SURE PROBE DOES NOT TOUCH STIR BAR - start collecting data - to reset probe, rinse it again, blot dry again, and PUT PLASTIC COVER BACK ON!

heart rate procedure

- use your index and middle fingers to count the number of beats for 15 seconds - multiply this number by 4, and this will be your BEATS PER MINUTE or bpm, the unit for heart rate - i am going to use the artery on my neck, called the carotid artery, make sure you know what artery you are using

nervous system of rat

1. Cerebral cortex - outermost layer of brain associated with highest mental capabilities 2. Cerebellum - controls balance, coordination, physical movement, and posture 3. Spinal cord - carries nerve signals from your brain to your body

appendicular skeleton of rat

1. Front limb - humerus, radius, ulna 2. Hind limb - femur, tibia, fibula

external anatomy of rat

1. Hair and claws 2. Body cavities - thoracic, abdominal, pleural (lungs), pericardial (heart) 3. Vibrissae - helps navigation in dark environments 4. Ear - Pinna (first part of ear that reacts to sound) 5. Eyes - Pupil and Iris - to see their environment 6. External nares - aid in breathing 7. Mouth and incisors and molars - mechanical digestion 8. External reproductive structures and openings (vaginal orifice, penis, mammary papillae, etc.) 9. Tail

excretory system of rat

1. Kidneys - filter blood, maintain concentration of water and salts in the body, form urine 2. Bladder - stores urine 3. Urethra - carries urine from bladder to orifice

respiratory system of rat

1. Larynx - air passage, voice box 2. Trachea - allows air to pass to and from lungs 3. Lungs - gas exchange 4. Diaphragm - contracts and relaxes during breathing

DNA Station order of reagents for PCR

Preparation of each PCR tube: First master mix, then human primer mix, then the DNA solution, then nuclease free water. REMEMBER: negative control does NOT contain DNA solution!

adaptations of angiosperms: fruits

Produced by angiosperms to attract seed dispersers

adaptations of angiosperms: flowers

Produced by angiosperms to disperse gametes (pollination) and create embryo (fertilization)

positive control purpose

made by staff with staff DNA, this control contains known template dna; used to show that the primers worked, that the master mix worked, and that the thermocycler worked (basically, that the amplicons were produced)

angiosperms: monocot vs. dicot

monocots have parallel leaf venation, and flowers in multiples 3. dicots have netlike leaf venation and flowers in multiples of 4 or 5. if it comes from woody growth (aka a tree) it is a DICOT!

bryophyte

nonvascular plant; examples are mosses and their relatives

Monocot stem

note that the vascular bundles are scattered

Monocot leaf epidermis

note the stomata note that the cells are arranged parallel

Dicot leaf epidermis

note the stomata note that the cells are arranged randomly

Dicot stem

note vascular bundles are in a ring-shape

Monocot root

note xylem and phloem are in a ring-shape

Elodea

notice the chloroplasts multicellular eukaryote (plant) photosynthesizes

Paramecia

notice the nuclei unicellular eukaryote (protist) does NOT photosynthesize

male cricket vs. female cricket

notice the ovipositor on the female, and generally females are larger

Dicot leaf

notice the various structures including upper/lower epidermis, spongy/palisade mesophyll

Diatom

notice their ornate silica cell walls unicellular eukaryote (protist - algae) photosynthesizes

using rat software

on the left menu in the software are all of the body systems listed out; all of them are visible/grayed out, so to find a specific organ first determine which system it belongs to then remove the others by pressing the system name until it is no longer visible you can drag the rat using your mouse by holding the left click, this helps with orientation to better see the particular organ.

Monocot leaf

only see vascular bundles

adaptations of gymnosperms/angiosperms: pollen

pollen is the haploid male gamete that can be delivered via wind or pollinators from one plant's flower/cone to another plant's flower/cone. this is a reproductive advantage for land plants

what is the purpose of the DNA wash buffers (pre-wash and g-dna wash)?

pre-wash buffer = removal of protein contaminants wash buffer = removal of salts and other contaminants prior to DNA elution. This ensures high purity DNA to be recovered

max absorbance at green

red emission

what is the purpose of the dna elution buffer/elute?

releasing of dna from the spin column matrix (dna elution = the process of extracting DNA from animal tissue samples by washing with a solvent)

pteridophyte

seedless vascular plant; examples are ferns

adaptations of gymnosperms/angiosperms: seeds

seeds are protective coats for a plant zygote and this is another reproductive advantage for these plants because the seed allows the embryo to survive for long periods of time before germinating (example: if conditions are too harsh, it can stay dormant in the seed)

what is the purpose of taq polymerase?

the enzyme that builds the DNA strand during PCR

DNA Station sizes of microcentrifuge tubes

the tallest jar = 1.5 mL medium jar = 2 mL small jar = 0.5 mL

Example electrophoresis diagram

this one has all students heterozygous, and no contamination has occurred because the far right well has no amplicons produced. the ladder can be seen at the far left (first well). remember, one band in a well means homozygous, while two bands means heterozygous. to determine how long an amplicon is, trace it back to the ladder to get an approximation.

what is the purpose of the nuclease-free water?

to dilute the concentration of the reagents to the proper final concentration. Also use of nuclease free water helps avoid DNA degradation by nucleases as well as interference of the PCR reaction by ions which could be present in otherwise not nuclease free deionized water.

what is the purpose of the 2x buffer?

to provide a suitable chemical environment for the activity of taq polymerase during PCR

cranial

toward head

medial

toward midline

caudal

toward tail

dorsal

towards back

ventral

towards belly

anterior

towards head/front

proximal

towards point of attachment

posterior

towards tail/back

to move the reticle unit ruler around

twist the eyepiece!

purpose of dna ladder?

use to identify band sizes of unknown amplicons from the known band sizes of the ladder

negative control purpose

used to detect any contamination of the PCR reagents with foreign DNA (you purposely don't add any student DNA to this control, and if there was contamination, you would see amplicons produced in the negative control well after gel electrophoresis)

angiosperm

vascular plant that produces flowers and fruits, examples are roses, lilies, trees, grass, etc.

gymnosperm

vascular plants that produce "Naked seeds", such as pine cones and other conifers.

max absorbance at purple

yellow emission

DNA Station order of reagents for buccal cell collection

Collection of buccal cells: add GLB (genomic lysis buffer), then add buccal solution with GLB into a spin column (small one) (which is in a collection tube (large one)), then add DNA Pre-Wash Buffer, then add g-DNA wash buffer, and finally add DNA Elution Buffer (elute).

purpose of 2% agarose gel?

it is what the DNA travels through

blood pressure procedure

- allowed to take off gloves here to better feel pulse of your LI, if you do then make sure to put ethanol on your hands to clean them real quick - make sure that LI is not sitting with legs crossed because that can interfere with blood pressure - you will place the stethoscope on your LI's brachial artery (right above elbow) - the units for blood pressure are millimeters of mercury, or mmHg - what matters in this procedure is more that you actually know how to take blood pressure than did you get an accurate reading - inflate the cuff to about 180 mmHg, then SLOWLY release the cuff and listen for the sounds and record the systolic over diastolic (the systolic will always be the bigger number)

beaker vs. graduated cylinder

- beaker (right), graduated cylinder (left) - can first pour out a large amount of algal solution from bottle into beaker - then pour from beaker into cylinder to get the precise amount needed

choice chamber setup for cricket animal behavior experiment

- can only have two choice chambers, and one cricket in the middle! - identify your independent and dependent variable and what factors you might control for (number of crickets, same item in choice chambers, amount of time per trial, etc.) - independent variable: food source (banana vs. apple for example) or could be presence/absence of light and you use a dark chamber, etc. etc. - dependent variable: food preference (time cricket spends in each chamber)

DNA Station know how to assemble and balance, then reset, casting unit

- first place the casting unit in a position where the large knob is toward the top away from you - then grab the tray and place it so the two handles are to the left and right - using the knob, lock the tray in place - now, grab the comb, and place it to the side closest to you (bottom) - grab the circular level and place it anywhere on the casting FRAME (NOT the tray) - use the three screws to center the ball on the circular level - to reset, take off circular level, take off comb, undo the knob, take off the casting tray, and clean everything

Transgenes/Enzyme Station prepare a cuvette with solution and use a spectrophotometer and Logger Pro to collect data

- grab a blank cuvette and pipette the given volume of correct colored solution (based on instructions) inside the cuvette - remember to use the right pipette for the volume asked, and the right tip, and to discard your tip after using it, and to close the bottle after using it - the cuvette has a little arrow on one side near the top, that arrow should be on the side of the arrow on the spectrophotometer when you place it inside - before placing cuvette into spectrovis (the spectrophotometer), make sure logger pro (on the computer) is reset and ready to collect data - once you place the cuvette into spectrovis, the computer should automatically create your max absorption graph

Anatomy Station skills/knowledge required to ace this section (any one of these could be assigned)

- know how to apply the anatomical terms such as anterior vs. posterior to a given practical situation - know how to use the rat dissection program on the computer to identify various given organs - know major cricket body parts/regions, and know how to identify the gender of a given cricket in a resin - know major rat body parts/regions as well as their functions, and identify a given organ on a rat under the fume hood - know organs and functions of the various systems of a rat that were studied in lab (slide 8 of lecture slides) and speak to their general functions - understand the evolutionary advantage/purpose of a rat's large liver, a male rat's large testes, and a female rat's uterine horn

DNA Station skills/knowledge required to ace this section (any one of these could be assigned)

- know how to assemble and reset gel electrophoresis rig - know how to assemble and balance, then reset, casting unit - know how to use a micro-pipettor properly to pipet some amount of DNA solution into a gel - know every single reagent involved during labs 11 and 12 (pre-pcr, pcr, and gel electrophoresis), their order, and the purpose of each reagent - understand the purpose of the controls and ladder, and understand how to interpret a gel electrophoresis diagram

Probes and Plants Station skills/knowledge required to ace this section (any one of these could be assigned)

- know how to prepare an algal solution of a given volume - recognize and know how/when to use different volumetric devices such as a beaker or a graduated cylinder - know how to set up a pH probe and how to use it to measure a solution's pH - know how to set up an optical dissolved oxygen probe and how to use it to measure a solution, including what units you are measuring in - know how to use a caliper to measure a given object such as a rubber cricket - know how to identify a land plant based on its macroscopic features (angiosperm, gymnosperm, etc.) - also know whether this plant specimen is a monocot or a dicot (IF GIVEN AN ANGIOSPERM) based on its macroscopic features - know what adaptations/features each type of land plant has (angiosperm, gymnosperm, etc.)

Transgenes/Enzyme Station skills/knowledge required to ace this section (any one of these could be assigned)

- know how to spread a bacterial culture over a plate and how to properly reset - know how to seal a plate with parafilm and reset - know how to properly label a bacterial culture plate after sealing it - interpret the three possible bacterial plates that we created during lab, and why they exhibit the genotypes/phenotypes that are observed - know how to prepare a cuvette with solution and use a spectrophotometer as well as Logger Pro to collect data - interpret a maximum absorption graph and describe what colors are absorbed and emitted for a given graph

Physiology Station skills/knowledge required to ace this section (any one of these could be assigned)

- know the proper choice chamber setup for the cricket animal behavior lab - know the proper chamber setup to measure the metabolic rate of crickets (co2 probe, odo probe, how many crickets in the chamber during experiment, etc.) - understand how a cricket's metabolic rate or gas production/consumption will be affected by changing the temperature and why - know how to take someone's blood pressure, and what blood vessel is used to do so - know how to measure your own heart rate and which blood vessel you are using

Transgenes/Enzyme Station LB + ampicillin

- this plate contains bacterial colonies - along with LB, this plate also contains ampicillin - the bacteria we observe in the colonies all survive because they have the BLA gene, which came from the PGLO PLASMID - the bla gene codes for ampicillin resistant - therefore all of these bacteria are TRANSFORMED/TRANSGENIC - transgenic because they contain the PGLO plasmid - genotype: LB and amp - phenotype: no fluorescing (b/c no arabinose present in the plate to express GFP gene)

Microscopes Station skills/knowledge required to ace this section (any one of these could be assigned)

- know the proper procedure to observe a slide under a light microscope (edge of coverslip, start at lowest objective, etc.) - identify what a given specimen(s) on a slide is by utilizing a light microscope properly - identify a cellular structure, multicellularity, and whether the specimen photosynthesizes (only if given elodea, diatom, or paramecium though cuz obviously the plant slides are all multicellular and photosynthesize, but still know major structures for plant slides as well) - identify whether a given plant specimen is a monocot or a dicot based on microscopic features observed using a light microscope - know how to measure a given specimen using a light microscope and convert from reticle units to micrometers (conversions will be given) - understand the difference between total magnification and objective (not as important for the practical, just for your own knowledge tbh) - KNOW HOW TO RESET YOUR LIGHT MICROSCOPE!! (it's like 3 points according to my LI so if anything do this and you get half the points already)

Transgenes/Enzyme Station proper labelling of a bacterial culture plate

- label on the BOTTOM OUTSIDE EDGE - include the following: - group number - LI name - lab room - day/time of lab - contents - EXAMPLE: Group 1, Jacob, 1.18, Thurs 6-10PM, GLO culture

Transgenes/Enzyme Station spreading a bacterial culture over a plate and proper resetting procedure

- obtain a plate with the growth medium inside - using a micro pipettor, pipet a given amount of the bacterial culture onto the center of the plate - remember to use the right tip, and discard the tip after use, and close all bottles after using them - from the brown paper bag, take out a bacterial spreader - spread the bacteria in a clockwise fashion, making sure to go at least one revolution - close the plate with its cap - PUT THE SPREADER INTO SOLID CHEMICAL WASTE - to reset, TAKE OUT SPREADER FROM WASTE AND PUT IT BACK INTO THE BROWN PAPER BAG!

setting up algal solution

- pour desired amount of algal solution into the graduated cylinder - put the stir bar inside the solution - put algal solution on top of stir plate and start stirring

DNA Station know how to use a micro-pipettor properly to pipet some amount of DNA solution into a gel

- put gloves on and clean with ethanol - remember to double check that you are using the correct pipettor - remember to double check that you are using the correct tip - grab the correct pipettor for the volume assigned - attach the correct tip properly (open tip box, push the pipettor onto a tip NEVER touch the tip with your hands, close tip box) - while holding pipettor to first stop, slide it into the given solution - slowly release the pipettor from first stop back to resting - slowly take out the pipettor from solution - close solution - while holding pipettor steadily with both hands and elbows rested on table for support, and holding pipettor vertically at all times, take pipettor to gel - slowly push pipettor into a well in the gel - slowly push pipettor to FIRST STOP ONLY - slowly take out pipettor and then release the stop back to resting - to reset, push pipettor tip into solid chemical waste, put pipettor back where you got it, clean table, clean gloves with ethanol

DNA Station know how to assemble and reset gel electrophoresis rig

- put the cover on the apparatus - literally just plug the red cable into the red one on the power supply and the black one into the black one lol - press start - to reset, press stop, take the wires out, take the cover off watch this video to refresh your skills: https://www.youtube.com/watch?v=o2wSL-G1IdE

proper procedure to observe a slide

- start at the edge of the coverslip, with the lowest objective, a comfortable light intensity, and the stage all the way up - bring stage down slowly and use the stage controls to find a hint/trace of your specimen - now you can zoom in more and use the fine knob to make things clearer

Transgenes/Enzyme Station sealing a plate with parafilm

- take an empty, closed plate - grab a piece of parafilm from the box and unwrap the seal - hold one end of the parafilm with one thumb, and grab the middle of the parafilm with the other hand's finger - keep gripping one end of the plate with thumb, and SLOWLY move the other end that is gripped in the middle of the parafilm across the plate

resetting algal solution

- take out the stir bar with magnet - pour the algal solutions in the cylinder/beakers into the chemical liquid waste bucket on table - using the dH20 bottle, rinse and dry the volumetric devices used

cricket metabolic rate experiment setup

- the O2 probe goes vertically, the CO2 probe goes horizontally, and the temperature probe is inserted horizontally facing DOWNWARDS - MUST HAVE 4 CRICKETS... NO MORE, NO LESS!!! - units for co2 output or o2 intake are in ppm - independent variable: time (seconds) - dependent variable: o2 consumption or co2 production, measured as the slope (ppm/s) - controls: same sample size of crickets, same temperature, same chamber, same sex of crickets, etc.

Transgenes/Enzyme Station interpreting a maximum absorption graph and listing what colors are absorbed and emitted for a given graph

- the maximum point on the graph indicates the wavelength at which the maximum light is absorbed for that particular solution - max point indicates what color is ABSORBED/TAKEN IN - you can use your knowledge of the color wheel to then figure out what color would be EMITTED/WE WOULD SEE - max point at orange = blue emission (and vice versa) - max point at green = red emission (and vice versa) - max point at purple = yellow emission (and vice versa)

setting up a pH probe

- the probe will be inside a yellow bottle labeled ph 7, this is to keep the pH neutral - take out the yellow bottle, cap it, then clean the probe by pouring some dH20 on it - grab a kim wipe and BLOT the probe dry (to prevent electrostatic charge and getting incorrect measurements) - now you are ready to stick the probe into your prepared algal solution (which should be sitting on stir plate being stirred) and start collecting data with logger pro - to reset, clean with dH20 again, blot dry with kim wipe, then put the probe back into the yellow pH 7 bottle

Transgenes/Enzyme Station LB only plate

- this is a bacterial lawn - only LB is present in the plate, which means that there are NO TRANSFORMED BACTERIA/no transgenic bacteria - the bacteria that are present in this lawn all survived the heat shock step - genotype: LB only - phenotype: no fluorescing (b/c there is no arabinose to express the GFP gene)

digestive system of rat

1. Mouth and Salivary glands 2. Pharynx, tongue, esophagus - carries air, food and fluid down from the nose and mouth; move food and assist swallowing; carry food and liquids from the mouth to the stomach 3. Liver - secretes bile, filters blood, digestive function (same color as spleen) they eat ANYTHING, their diets are vast and to make up for it, they have big livers 4. Stomach - mechanical and chemical digestion 5. Small intestine - duodenum, jejunum, ileum; digestion of food and absorption of nutrients 6. Pancreas - pancreatic juices to help with digestion 7. Cecum - bacteria for plant digestion; junction btwn small and large intestines 8. Large intestine - absorbs water and forms feces

male reproductive system of rat

1. Scrotum - contains testes 2. Testes - sperm is stored here; male rats have huge testes which is a reproductive advantage because more sperm is stored which means more chances of having offspring and ensuring survival/fitness 3. Penis - urine and semen ejaculation 4. Epididymis/vas deferens - collection/storing (epi) and transportation (vas) of sperm

axial skeleton of rat

1. Skull 2. Vertebral Column

immune system of rat

1. Spleen - helps to destroy old red blood cells, form lymphocytes, and store blood

circulatory system of rat

1. The Heart - know atria, ventricles (right atrium, top left, right ventricle, bottom left, left atrium, top right, left ventricle, bottom right). 2. Veins: anterior and posterior vena cava (carry blood from body to right atrium, anterior carries blood from head whereas posterior carries blood from bottom region) 3. Arteries: aorta (largest artery, remember arteries carry blood away from heart and veins toward heart)

female reproductive system of rat

1. Uterine horn - the point where the uterus and fallopian tube meet; its length and shape allows several fetuses to develop simultaneously which is a reproductive advantage 2. Ovaries - egg production

DNA Station order of reagents for gel electrophoresis

2% agarose gel, syber-safe dna stain DIRECTLY INTO GEL, then IN THE DNA SAMPLES put 6x DNA loading dye solution, then pour TAE buffer OVER THE AGAROSE GEL, then pipet the DNA samples into the wells IN THE GEL

purpose of syber-safe dna stain?

allows you to see the location of the DNA amplicons (it is a dna stain that binds to the amplicons)

lateral

away from midline

distal

away from point of attachment

what is MgCl2 and its purpose in PCR?

its is a cofactor that enhances the activity of taq polymerase, boosting amplification during PCR

max absorbance at orange

blue emission

what are dNTPs?

building blocks of DNA, these are single nucleotides (monomers), stands for deoxyribonucleotide triphosphate

purpose of TAE buffer?

carries the electrical current through the gel

direction of amplicon migration during gel electrophoresis

cathode (negative end) to anode (positive end); aka black side to red side

adaptations of vascular plants: vascular tissue

consists of xylem and phloem, which transport water, nutrients and sugar throughout the plant. this allows the vascular plants to live in a variety of environments

what does genomic lysis buffer do?

disrupts cell membranes of buccal cells

purpose of 6x dna loading dye solution?

dna is colorless, so adding the loading dye helps to determine the rate of movement of different size protein molecules in the gel during electrophoresis. Examples of loading dyes that move with the DNA sample include bromophenol blue.

crickets are? what does this tell someone about a cricket's metabolic rate under various conditions?

ectotherms; therefore, their metabolic rate is directly related to the surrounding temperature (an increase in temperature corresponds to an increase in metabolic rate/respiration and so we expect more oxygen consumption and more carbon dioxide emission)

what is the purpose of the human primer mix?

it contains the forward and reverse primers specific to the DS180 locus; the primers flank the target sequence and are the starting points of DNA synthesis