Bio II: Genetics and Evolutionary Biology - Genetics Lab | BIO 1022, Lab Reports of Biology

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Bio 1022/25 Bio II: Genetics Lab

Lab Manual

Spring 2008

Preface p. 2

Saint Joseph's University

Bio 1022/5 Biology II: Genetic & Evolutionary Biology

GENETICS LAB

Class Information:

‡ Class bulletin board: in the a hall between 212 & 214 SC across from the main office (229 SC) ; Pick up corrected assignments, handouts, corrected quizzes from the bins on the wall beneath the bulletin board. Please respect others' privacy. Note which are turn-in and which are pick-up!!  Genetics Lab –general info, links, announcements & reminders www.sju.edu/biology/genetics.htm

Bio 1022 Genetics Lab Section List Spring 2008 5 Section 153 MONDAY Dr. Ratterman 212 SC 2:30 – 5:30 pm 5 Section 152 MONDAY Dr. Coffey 214 SC 2:30 – 5:30 pm 5 Section 151 TUESDAY Dr. Ratterman 212 SC 2:00 – 5:00 pm 5 Section 156 TUESDAY Dr. Lee 214 SC 2:00 – 5:00 pm 5 Section 157 THURSDAY Dr. Ratterman 212 SC 2:30 – 5:30 pm 5 Section 154 THURSDAY Prof. D’Angelo 214 SC 2:30 – 5:30 pm 5 Section 155 FRIDAY Dr. Ratterman 212 SC 2:00 – 5:00 pm Bio 1025 332 WEDN Eve Prof. D’Angelo 214 SC 6:15 – 8:55 pm

Your professors:

Core Lab Coordinator: Dr. Denise Marie Ratterman Office: Rm 226 SC or look for me in the Core labs 212 or 214 SC if not in my office Also be sure to check my preproom 201A behind 200 SC and at the photocopier in 229 SC Office Hours: M-F 10-11 AM or sign up for other times on my door signup sheet x1822 on campus or (610)-660-1822 from off-campus cell 610-308-6905 (text/voice)  email [email protected]  www.sju.edu/biology/dratterm.htm Available for questions in LaFarge Science Hall at night: Rm 400 LaFarge Sun-Thurs 10:30 pm-12:30 am (after midnight) x You don't have to be in one of Dr. R's sections to ask her a question...

BIO 1021/1022/1025 Lecture & Lab Instructors: Dr. Michael McCann 112E B/L x1823  [email protected] Lec Sec151 in 300SC Dr. John Tudor 223 SC x1821  [email protected] Lec Sec152 in 200SC Dr. Christina King Smith 222 SC x1118  [email protected] Lec Sec153 in 109SC Dr. Caroline Coffey 108A SC x1931  [email protected] Lec Sec154 in 101SC Dr. Denise Marie Ratterman 226 SC x1822  [email protected] Dr. Julia Lee 227 SC x3439  [email protected] Prof. Louis D’Angelo 313A SC x3076 or leave a message at x1820 or 229SC

 Faculty web pages can be accessed via www.sju.edu/biology/ Main Biology Office: Mrs. G Rm 229 SC x1820 on campus or (610)-660-1820 from off-campus

This manual belongs to: _____________________________________________

If found, please contact me at: _______________________________________(phone or email)

Preface p. 4

GENETICS LAB course schedule grid by lab up to break Spring 2008

Week of: Lab 1 Lab 2 Plant F

Lab 3 Drosophila

Lab 4 Bacteria Events Jan. 14- MONDAY - FRIDAY

1a: Mitosis 2a plant seeds

Jan. 23-25,28, W ED-FRI , MON,TUE

1b: Mitosis & Meiosis

1 week observations (or 2 week counts)

3a observe flies learn sexing

Jan 30-Feb 1, 4, 5 W ED-FRI , MON,TUE

2b: corn F kernel analysis (3 wkshts)

2c: 2 or 3 week counts Feb. 6-8, 11, 12♥ W ED-FRI , MON,TUE

2c: 3 week observations

due: report draft check

Come in and clear fly vials in the morning before your lab

Visiting researcher talks about Drosophila

3b: set up reciprocal rosses (PxP mutant x wild) Feb. 13-15, 18, W ED-FRI , MON,TU

Covered on practical

Due: Plant F lab report

Covered on practical

3c: Clear PxP vials

Practical I (labs 1 & 2)

Feb. 20-22, 25, 26 W ED-FRI , MON,TU

3d: Drosophila F1 counts, set up F1 x F1 crosses

due: F predictions (quiz) Feb 27- W ED-FR BEFORE BK

3e ( clear F parents)

Wed- Fri: Prelim analysis F2 predictions due (oral quiz)

Spring Break March 3-

Dr. R clears for Mon & Tues

Preface p. 5

GENETICS LAB course schedule grid by lab after break Spring 2008

Week of: Lab 1 Lab 2 Plant F

Lab 3 Drosophila

Lab 4 Bacteria

Events

March 10, 11 MON, TU AFTER BK

Mon & Tues Prelim analysis /F2 predictions due (oral quiz) Mar 12-14, 17, W ED-FRI , MON,TU

3f: count F offspring

Fly lab draft chk ( F1 parts) Mar 20-21, 24 Easter No lab Thur-Mon Mar 25-28, 31 TUES -FRI , MON

4a: Aseptic Techniques Apr 1-4, 7 TUES -FRI , MON

Due: Drosophila F1 & F2 Lab Report

4b: Bacterial Conjugation

Apr 8-11, 14 TUES -FRI , MON

Lab 4c: DNA Plasmid Isolation 4d: DNA Gel Due: Bacterial problems Apr 15- TUES -FRI

Genetics Jeopardy Monday invited to any of the games

Apr 21- MONDAY - FRIDAY

Due: Bacterial Conjugation Lab Report Includes gel standard curve

Covered on practical

Practical II (labs 3 &4)

Preface p. 7

Students will learn this from demonstration and explanation of techniques/approaches by the instructor and in the lab manual, actually carrying out the procedures in lab with their teams over many weeks, and worksheet and practice problems for the dilution and plate count calculations. Each student will test their aseptic technique for transfers using a control and one strain. Each student will calculate bacterial concentrations for their team’s plate counts from standard and selective/color indicator agar and report these values in their bacterial report.

Students will be assessed through a practical exam and other assignments. The practical exam includes having the student demonstrate their ability handle bacteria properly (aseptic technique) as well as questions relating to the techniques, calculations, and interpretation of data. Other assignments include worksheets with colony count/dilution calculations and a final lab report on the bacterial work.

Grading: The lab grade is 25% of the total course grade : (lecture average as %) (0.75) + (lab average as %) (0.25) = overall course average % see lecture syllabus for weighting of quizzes/homeworks/exams/final in lecture average and course grading scale

Your running lab average can be calculated at any time: (Your current point total) x 100% = Lab average as % (total possible points for those assignments)

ASSIGNMENT TOTAL POSSIBLE POINTS Three lab reports 100 pts. each 300 pts. Two lab practicals 100 pts. each 200 pts. Worksheets 10-20 pts each 85 pts Quizzes (Written / Oral) for Fly Lab 30 pts Labwork Checks (work outside of class done?) 25 pts Draft Checks 5-10 pts each 20 pts Spot Checks (lab notebook 20 /topic sheets 20) 40 pts. Total 700 pts.

Attendance: Students are expected to attend all labs. You should attend only your scheduled section (Monday, Tuesday, etc); crossover is not allowed. Unexcused absences will result in a zero for that lab's report or that day's exam. At the instructor's discretion, make-up dates can be arranged for excused absences; you must talk with your instructor before coming in to make up - because of the timing of team work / observations make-ups may not be possible for some labs. Please note that same lab is run Th, F, then Mon, Tues until after Easter. Lab practicals are set up during one week only (Practical I Th/F then following Mon,Tu; Practical II Mon-Fri in same week). It is your responsibility to contact your instructor (or Dr. Ratterman, if necessary) as soon as possible. Two unexcused lab absences will result in a FA for the entire course.

Calculators: Handsprings, Palm Pilots, hand-held computers, and graphing calculators are not allowed at practicals. Buy a cheap calculator to bring with you!! You need only simple functions.

Questions & Concerns: ASK!!! Do not let anything stop you from asking. Ask for help when you start to fall behind or do not understand an idea or calculation.

Academic Honesty Policy: Cheating on a lab exam will result in a zero for that exam. Plagiarism in written assignments will result in a zero for that assignment. Teams & sections will share data but each individual is expected to author his/her own lab report (including doing any calculations and library research yourself). Do not use old lab reports, even as an aid, as this causes noticeable inconsistencies in reports. You should author your report originally yourself. Any student caught cheating for a second time will receive an F for the course. All incidents of cheating will be reported to the SJU Board on Academic Honesty. Do not copy even one sentence from your text, manual, or any published work.

Preface p. 8

Lab Reports - See the end of each lab and appendix for additional guidelines:

1. Lab reports should be typed double-spaced and conform to the text page limits. Text page limit includes Abstract through Discussion and is 9 pgs for lab reports. Your instructor will not read past this page cut-off. Condense your report and adjust print out so that it fits this limit. Tables & Figures are not in page count. Literature Cited, Tables, Figures, and Calculations pages should be placed after the text. Choose a readable size font; if it is too small your instructor may require a reprint that conforms to the page limits. Black text or other readable color is acceptable. 2. Three references are required. Your class text can count as one of these sources, but your lab manual does NOT count as one of the three. No encyclopaedia as sources. Only one of the required three can be a web source. Note: only some web sources are appropriate (NOT teacher notes online). You need a full citation for any web source. Some materials will be on reserve at the library. Check the information you state against the information in your source for accuracy -- do not cite a source for information not actually found in that source. 3. Start early. In genetics lab, data will be collected over several weeks. Don't wait until all is finished to start your report. Start it early and add to/revise to lighten your time burden and keep you connected. Turn in your report at the beginning of your regular lab period on its due date. 4. Keep an electronic copy of your text, tables and graphs, a photocopy of hand-written pages, and your original copy of the raw data. Always leave your original data bound in your lab notebook. In the event of a lost report, you can then produce a new copy easily and quickly. When working on a word processor, save your file often! Save it before any difficult operation such as spell-check, inserting symbols, or printing. Change the name slightly to save a new draft without copying over an old draft before major changes so if all is lost a recent old draft is still accessible. Print out a draft. Never leave your only copy on someone else's hard drive -- save it to a diskette, USB drive, CD or email a copy to yourself. Keep diskettes/USB drives free from harm, high/low temp and magnets. When in doubt, always back up your files again.

Electronic submission: Your instructor may try electronic submission & grading of reports, if so, additional guidelines will be given out. Unless directed as such you should NOT try to submit their reports electronically, a hard copy is required.

Late Reports: Late reports and assignments will be docked late points as determined by your section's instructor. Turn in late papers as soon as possible, do NOT wait until your next lab meeting. Excuses will not be granted for computer problems, so print out your report early and keep a second diskette, CD, email, network and/or hard copy of your report in progress in case your computer’s copy is lost or corrupted. It is your responsibility to find out how to hand in your late report -

• instructors have different policies regarding this. No reports will be accepted more that 1 week late or any later than Wednesday of Finals week (4/30/08), whichever comes first.

Grades, Scores, and Assignment Pick-up: SJU policy does not allow the posting of individual grades because of privacy issues. Lab reports and assignments are often handed back during lab class; however, we have found it very useful and convenient for students if some assignments can be picked up from the table outside of Rm 212 (next to the Genetics Lab bulletin board). Dr. Ratterman places "bins" for pick-up of corrected assignments on this table for lab and lecture. Please be considerate of the privacy of other students when picking up your papersIf you request that your own lab assignments be held for your own pick-up rather than placed out for pick-up, we would gladly honor your request.

Computer Accounts: If you already have a computer account, make sure you know your login and password. If you are still using the issued password, it is time to change it !! Change your password every so often to protect your account. Change it via the web at my.sju.edu.

Here are example pages of how your lab notebook might look: From “DR’s” notebook… DR has teammates AS and TJ

1/02/03 Lab: Test of True-breeding for stocks to be used in a complementation exp

23 ˚C Strain: mutant 1

8:30 AM Cleared: 8:30 AM 2 vials, placed on bench procedure p.

1/02/03 Observation of non-virgin stocks of mutant 1

23.5 ˚C immobilized on ice dissecting scope 20x procedure p. 25-

2:30 PM Both males and females had dark areas looking like a black powdering on their dorsal sides. The thorax was particularly dark on the dorsal region. Banding on the dorsal side of abdomens of females was still able to be seen as black bands. Ventral side of the abdomen was lightly-colored similar to wild. Next to wild on white plate they appeared darker and more grey, the wild appearing more tan or brownish in contrast.

(head) Darkest here No stripes (thorax) drawing of female mutant 1 (legs & wings not shown) (abdomen) stripes

Set up of cross 3 pm from stocks cleared 8:30 am 4 virgin ♀ mutant 1

2 virgin ♂ mutant 1 (one squashed of original 3)

Vials prepared as on p. 30-31 0.5% propionic acid used Flies held on ice for less than 9 min gender confirmed with hand lens and then checked for sex combs w/ dissecting scope at 20x

3:30 pm check: all 6 flies up and about. One female looks like it has a bent wing but is moving around okay. Vial placed on tray on side bench

overhead lights kept on 2 4 h /day

Preface p. 10

1/03/03 Check of cross vial next day:

23 ˚C All six flies moving around. The one female still has a bent wing.

One of the males was holding his right wing sideways (think he is courting with song) when observed.

1/09/03 Clearing of parents:

21.5 ˚C Cross vial cleared –all adults removed to morgue (p. 28,31) DAY 7 for cross

A small amount of 0.5% propionic acid added since top looked a bit dry Some larvae present but are very small Placed it back in same spot

1/16/03 Check of cross on regular lab day:

23 ˚C Pupae but no adult flies yet in vial DAY 14

1/17/03 F1 counts: have wild and mutant 1 out for comparison

23 ˚C

Mut 1 x mut 1 F1 1/17/ (15 d)

(18 d)

TOTAL

dark body males 23 12 18 59

dark body females 18 15 17 5 4

Wild-type males 0 0 0 0 0

wild-type females 0 0 0 0 0

Collected by DR AS TJ AS* DR TJ *rechecked AS’s notes, was 6 males, 4 females not other way

None looked wild could tell phenotypes without lens on white plate

Subcounts ♀ 12 +6 = 18 ♂ 18+5 =

1/19/03 F1 counts DR DAY 17

23 ˚C

dark body MALES - NOTE: these added to table above

dark body FEMALES note: 2 flies newly-emerged and very white -- put back in cross vial (not counted today)

prediction P: dd (dark) x dd (dark) F1: all dd (all dark) d d d dd dd

d dd dd

Preface p. 11

St. Joseph's University - Biology II: Genetics

LAB #1 - MITOSIS and MEIOSIS

Lab 1a: MITOSIS

The Cell Theory

1. All organisms consist of one or more cells
2. The cell is the basic unit of structure for all organisms 3. All cells arise only from pre-existing cells

Cell Division

In today's lab we will study the process of cell division in eukaryotic cells. During the process of mitosis , one cell divides to form two daughter cells. Both daughter cells receive an identical copy of the genetic information. The genetic information contained in the nucleus is replicated prior to the actual division of the cell. The separation of the genetic material is referred to as karyokinesis (nuclear division). The process of dividing the cytoplasm is referred to as cytokinesis. In lab, you will watch a time-lapse movie of a cell dividing by mitosis. The chromosomes (carrying the cell's genetic information), align at the center of the cell and are separated in an orderly manner toward the two sides (two "poles") of the cell.

Cell Division in Early Development

After fertilization of an egg (ovum) by a sperm, the offspring produced is a single- celled zygote. Subsequent cell divisions increase the number of cells into what, at first, looks like a ball of cells. This is the morula stage. Subsequent changes result in the blastula and other stages of embryo development. In lab we will view a time-lapse movie showing early development of an embryo. Cell division occurring during early development is referred to as cleavage ; but, the process is still mitosis. Each cell in the embryo contains a full set of the genetic information as was present in the single-celled zygote. The micrograph below shows starfish embryos in these early stages.

Lab 1: Mitosis & Meiosis Lab p. 1

Mitosis

Next, teams will use the microscopes at their benches to view cells that have been stopped while in the process of dividing. These sections of dividing tissue have been stained so that the chromatin/chromosomes are stained darkly (usually purple or black in color). When you look at these cells remember what you see is a moment in time and that mitosis is a dynamic process. You can see in your mind's eye the stages of mitosis in motion.

The four stages of the process of mitosis are called PROPHASE, METAPHASE, ANAPHASE, and TELOPHASE. Use the diagrams to note the position and appearance of the chromosomes in each phase. In anaphase and telophase, the chromosomes will appear in two distinct groups within the same cell. In prophase, you may only see some "squiggles" of chromosomes within the oval or rounded nuclear area. Read over the description of what occurs during each of these phases in your textbook. Can you identify the spindle? During what phase will division of the cytoplasm (cytokinesis) be apparent?

There are examples of both plant and animal mitosis in your sample box: onion root tip (the genus name is Allium, this may on the slide label) and whitefish blastula. These are specimens in which many of the cells are undergoing mitosis. The root tip, for example, is an area of rapid growth and many new cells are formed. In seed plants, areas of cell division are called meristems. The "blastula" sample is that of an early stage of development of the whitefish. The blastula provides a good sample for the study of mitosis because of the active cell division at this time in development.

Observe both the onion and whitefish slides carefully. Can you see chromosomes in some of the cells? Look closely. What do the chromosomes look like? The chromosomes are easiest to see in the onion because they are very large; however, if you look for small X or V shaped, darkly-stained bodies you will find them in the whitefish sample as well.

Find an onion cell and a whitefish cell in each of the four stages of mitosis: PROPHASE, METAPHASE, ANAPHASE, and TELOPHASE. Draw a picture of each. Your team can work together to find cells in each phase; make sure each of you look at each sample and can find cells in the different stages of mitosis. Draw a picture of one cell in each phase in your notebook for both onion and whitefish samples. Draw pictures from looking at the cell through the microscope -- not looking at your teammates paper! Look for the mitotic apparatus (spindle & centrioles; note where the centrioles would be located --the centrioles are too small to actually see) Take note of what you see. Also make a note of what you cannot see that you looked for. In what ways are the two samples similar? different?

Lab 1: Mitosis & Meiosis Lab p. 2

A

C

B

Figure 2. Micrographs of whitefish blastula.

A. Section of a slice of an embryo. On our slides the section may be pink or violet and the chromosomes a darker violet or blue violet. Find a dividing cell with a visible spindle to help you begin to see the chromosomes.

B. The interphase cells may have a very light or non-visible nucleus. When the chromosomes are condensed enough to be seen this would indicate prophase.

C. Example of a telophase cell in whitefish. These are rare in samples. Look for remains of the spindle to help indicate this is telophase rather than two touching prophase cells.

Lab 1: Mitosis & Meiosis Lab p. 4

Interphase

Some of the onion root tip cells were not undergoing mitosis at the time the specimen was fixed and stained. These cells have a defined nucleus that, overall, stains purple but does not have visible chromosomes. It may have one or more darkly- staining circular regions; these are the nucleoli (nucleolus - singular; nucleoli - plural). When the cell is not in mitosis, it is in INTERPHASE. In the whitefish slides, the interphase nuclei do not stain much more than the other areas of the cell and so you will see no distinct nucleus. Only when the chromosomes are more condensed (during prophase) do these samples seem to show more darkly-staining areas in the nucleus.

The Cell Cycle

The pie diagram that depicts the Cell Cycle (Your textbook Fig. 1.14) shows what happens to a cell that is actively dividing. After one round of division, the newly-formed cell enters G1 phase. Later, it enters S phase. Then, G2 phase. G1, S, and G2 are all part of interphase. The cell leaves G2 as the process of cell division (mitosis or meiosis) begins. An important area of cell biology is the study of the control of the cell cycle to understand what signals tell a cell to divide or to cease dividing. Listen in lecture for key information about what happens in the phases of the cell cycle. During which part does replication of DNA occur?

Karyokinesis and Cytokinesis

Take a look back at the whitefish and onion samples keeping in mind that not only karyokinesis (nuclear division) but also cytokinesis (division of the cytoplasm) occurs during cell division. Find an onion cell and a whitefish cell in which you see evidence of cytokinesis and draw/describe. There are differences in the cytokinesis of plant and animal cells. Which one has a cell plate that starts to forms in the center of the cell? Which one has a contractile ring that forms, appearing to "pinch in" at the sides? Look at the cytokinesis electron micrographs at the demo station to see examples of each type.

Multinucleate Cells

Most nuclear divisions (karyokinesis) are followed by the division of the cytoplasm (cytokinesis) and the separation of two new cells. However, there are examples where this does not happen. Multinucleate cells are observed in a number of instances, such as in some types of algae and fungi, in many liver cells, in the developing embryo sac and endosperm of plants. Observe the demo slide of liver. See if you can trace find a binucleate liver cell with two purple-stained nuclei within it (note: nucleus - singular, nuclei - plural).

Lab 1: Mitosis & Meiosis Lab p. 5

Spermatogenesis

Examine the diagrams of spermatogenesis at your team bench. Note the difference between a primary spermatocyte, secondary spermatocyte, spermatid, and spermatozoan. How many sperm are produced from just one primary spermatocyte undergoing meiosis? If the testes produce the sperm from the meiosis of the spermatocytes, then why doesn't the male eventually run out of available cells? If the animal is diploid: which of these cells are diploid? haploid?

Gamete formation involves not only meiosis but also a maturation process to form the mature gamete. In spermatogenesis, this usually involves the development of spermatozoa that have a distinct tail. There is a diagram of this process at the demo table. Mature sperm can appear quite different in different species. Compare the bull sperm and frog sperm at the demonstration station.

Look for the spermatocytes, spermatids and spermatozoa in the samples of the rat (mammal) and grasshopper (insect) testes (Table I).

Table I. Guidance for examination of testes slides and diagrams. Testes of NOTES rat (mammal) Each seminiferous tubule is seen in cross section. Look at the outermost ring of cells: these are the spermatogonia. As you move in toward the center of the tubule you can view the subsequent stages of spermatogenesis: primary spermatocytes, then secondary spermatocytes, farther in, spermatids, and, in the center, spermatozoa with distinct heads & Tails

.

Lab 1: Mitosis & Meiosis Lab p. 7

Testes of NOTES grasshopper (insect) In these cross sections, you can view the different stages as you move from one end to another in each compartment. Spermatocytes are large and round. Spermatids are more elongate. In spermatozoa, the sperm heads are so long and thin that the groups of mature sperm look like bunches of hair!

There are some labeled diagrams at your team bench to help you identify the stages. Do not worry about distinguishing between primary and secondary spermatocytes in samples, but understand what the difference is, in terms of meiosis.

Testes of NOTES Crayfish testes (Crustacean)

In the rat and grasshopper testes samples, you may be able to distinguish the different stages, but it is rare to be able to recognize the phase of meiosis in a cell dividing in meiosis. To look for phases of meiosis, examine the crayfish testes slide.

Can you find a cell that is in Prophase I or II? Metaphase I or II? Anapase I or II? Telophase I or II? The spermatozoan in crayfish is actually star-like in shape. Can you find any?

Lab 1: Mitosis & Meiosis Lab p. 8