practice in genetics lab report

Using a forceps to pick up onion root samples preserved with Carnoy solution in falcon and place them in the watch glass.. Remove all remaining water from the watch glass with a pipette,

VIETNAM NATIONAL UNIVERSITY – HCMC

INTERNATIONAL UNIVERSITY

PRACTICE IN GENETICS

LAB REPORT

Group No.4 Group members:

Course name: Practice in Genetics (S1_2023-24_G03)

Instructor name: Dr Thong

TABLE OF CONTENTS

REPORT 1: MITOSIS 4

1 Introduction: 4

2 Procedure: 4

3 Result: 5

4 Discussion: 7

5 Conclusion: 7

REPORT 2: MEIOSIS 8

1 Introduction: 8

2 Procedure: 9

3 Result: 10

4 Discussion: 12

5 Conclusion: 13

REPORT 3: EXTRACTION OF DNA FROM PLANT CELLS 14

1 Introduction: 14

2 Procedure: 14

3 Results: 15

4 Discussion: 15

5 Conclusion: 16

REPORT 4: DNA QUALITY AND QUANTIFICATION 17

1 Introduction: 17

2 Procedure: 17

3 Results 18

4 Discussion: 18

5 Conclusion: 19

REPORT 5: CULTURING AND EXPERIMENTAL MATING OF DROSOPHILA MELANOGASTER 20

1 Introduction: 20

2 Procedure: 20

3 Results: 21

4 Discussion: 22

5 Conclusion: 22

REPORT 1: MITOSIS

1 Introduction:

As we have known, mitosis plays a very important role for the growth of organisms as well as repairing damaged tissue Mitotic cell division is the process that creates two genetically identical daughter cells from a single mature cell Mitotic cell division is divided into two steps: mitosis (nuclear division) and cytokinesis (cytoplasmic division)

When mitosis occurs, chromosomes are duplicated and separated into the opposite ends of the cell After that, the cytokinesis takes place when the nucleus is formed in each daughter cell as well as the division of cytoplasm

Mitosis consists of four phases: prophase, metaphase, anaphase and telophase

- In prophase, the chromosomes are condensed, the nucleolus disappears, the nuclear envelope begins to break down and the centrosomes migrate toward two opposite poles

of the cell Spindle microtubules form and bind to chromosomes at kinetochores

- During the metaphase, the chromosomes are arranged at the equatorial plate of the cell

- At anaphase, sister chromatids are separated and move to the opposite poles of the cell

- Telophase takes place when the separation at anaphase is completed At this moment, chromosomes relax, nucleolus and nuclear membrane begin to reappear and the spindle apparatus disintegrates Cytokinesis occurs, separating the two nuclei into two cells

The objectives of this investigation are helping students understand the sample preparation procedure before observation, as well as the main events of mitosis in onion root tips

2 Procedure:

Materials:

First, necessary materials and equipment are needed to be prepared:

- Onion root tips (preserved in

Procedure to prepare sample on microscope slide and observe:

1 Using a forceps to pick up onion root samples preserved with Carnoy solution in falcon and place them in the watch glass

2 Cut off 2 - 3 mm of the onion root tip as a sample and wash with water three times

3 Remove all remaining water from the watch glass with a pipette, add a few drops of 1M HCl solution and gently heat the watch glass a few times over an alcohol burner Cover the solution with a petri dish and wait for 10 minutes

4 Wash the onion roots again with water three times, then drain the remaining water from the watch glass and continue to add a few drops of orcein stain to the samples

5 Continue to gently heat the watch glass over the alcohol burner and wait for about 10 minutes

6 Use forceps to remove the sample from the stain solution and gently place it on the microscope slide

7 Put a few drops of glycerin on the sample, cover gently with coverslip, avoid air bubbles appearing under the coverslip

8 Gently tap on the coverslip surface to spread the onion root sample thinly

9 Observe the sample under a 10x microscope to determine the area where the cells are dividing Then gradually increase the magnification of the microscope to determine the division phases during mitosis of onion root cells

3 Result:

Draw and clearly label cells in various mitotic phases that you observe

All cell samples were observed under a microscope with 40x magnification

Prophase

Metaphase

Anaphase

Telophase

4 Discussion:

The use of Carnoy’s solution:

Carnoy's solution is a mixture of absolute alcohol, chloroform, glacial acetic acid, and ferric chloride As a pretreatment solution, Carnoy's solution is utilized Absolute alcohol shrinks the tissue, which causes it to stiffen, glacial acetic acid increases tissue swelling and inhibits overhardening, chloroform accelerates fixing, whereas ferric chloride acts as a dehydrating agent Carnoy's solution is used as a fixative since the purpose of soaking onion root tips in it

is to fix the DNA of the root tip cells

The purpose of adding HCl 1M solution and heating the sample over the alcohol burner:

HCl 1M was used to soften the root tips so that the stain can easily go into the cells and the onion root cell can be easily stained

The purpose of heating is to accelerating the reaction speed, making the stain penetrate into the cells

5 Conclusion:

After the class, students had the opportunity to strengthen their skills in using the microscope and learn more about sample preparation and handling procedures before observing onion root cells Furthermore, the observation also helps students distinguish and recognize phases in mitosis During the experiment, some operations using the microscope were noted as errors and will be learned from and practiced more carefully in the following practice sessions

REFERENCES:

[1]How does Carnoy fixative work? – TeachersCollegesj (2020, May 31)

https://teacherscollegesj.org/how-does-carnoy-fixative-work/

REPORT 2: MEIOSIS

1 Introduction

Gametes, specialized for sexual reproduction, are haploid cells carrying a single set of chromosomes and genetic information They're formed through meiosis, a cell division process reducing chromosome number from diploid to haploid This reduction is crucial for restoring diploidy upon fertilization when two gametes merge Meiosis involves an initial interphase where DNA replicates, followed by two rounds of division (meiosis I and II), each comprising prophase, metaphase, anaphase, and telophase Between these rounds lies "interkinesis," during which the nuclear membrane reforms, the spindle breaks down, and chromosomes relax

Meiosis involves the following phases:

- Telophase I: Chromosomes reach spindle poles, cytoplasm divides, but chromosomes

do not completely uncoil

- Prophase II: Chromosomes decondense, spindle reforms, and the nuclear envelope breaks down again

- Metaphase II: Individual chromosomes align at the plate, with sister chromatids facing opposite poles

- Anaphase II: Sister chromatids' kinetochores separate, pulling chromatids to opposite poles, now each chromatid is a distinct chromosome

- Telophase II: Chromosomes reach spindle poles, nuclear envelope reforms, cytoplasm divides, and chromosomes relax

The objective of this investigation is to describe the main events of meiosis, identify cells in garlic chive flowers at meiotic phases, and compare and contrast meiosis with mitosis

1 Use forceps to transfer garlic chive flowers to a watch glass, use dissecting needles

to remove perianth and petals to retain anthers

2 Wash the anthers with water 3 times Completely remove the water

3 Add several drops of 1N HCl solution into the watch glass, gently heat the watch glass over an alcohol burner for a few seconds Cover the watch glass with a petri dish and wait 30 minutes

4 Wash the anthers with water 3 times Completely remove the water

5 Add several drops of aceto-orcein stain into the watch glass, then gently heat the watch glass over an alcohol burner for a few seconds Repeat the step of adding aceto-orcein stain and heating 2-3 times, wait 5 minutes between each time

6 Transfer four stained anthers to a microscope slide, add one drop of 10% glycerol Cover with a coverslip

7 Use the handle of the dissecting needle to gently apply pressure over the coverslip This should squash the anthers into a thin cell layer

8 Observe the specimen under a low power objective to identify the area with many dividing cells Then use a higher power objective lens to carefully observe cells in different meiotic phases

Telophase I

Prophase II

Metaphase II

- Synthesis of DNA is occured

- Taking place in cell nuclei

- New cells are created

- Both follow the same steps which are prophase, metaphase, anaphase and telophase

- Begin with one parent cell

Differences:

- Occurs in all organisms (except

viruses) - Occur in animals, plants and fungi

- Involving all cells (both somatic and

germ cell)

- One round cell division

- No recombination or crossing over

between chromosomes

- Produce two diploid daughter cells

- Daughter cells are identical

- Function: cell growth and repair, cell

division

- Involving germ cells

- Two round cell divisions

- Occur recombination/crossing over between chromosomes

- Produce four haploid daughter cells

- Daughter cells are genetically different

- Function: contribute to genetic diversity through sexual reproduction

Garlic chive flower size affects the ability to find meiotic phases:

We repeated the practice of collecting buds in 4 flower samples: 2 old flower samples and 2 young flower samples and observed In younger flower samples, the rate of finding meiotic phases was higher than in the remaining samples This may be explained by that older buds are usually past active meiosis and younger buds are more likely to have cells undergoing meiosis

5 Conclusion:

Following the lesson, students could hone their microscope abilities and get knowledge about sample handling and preparation techniques before looking at garlic chive flower cells Additionally, the observation aids students in differentiating and identifying meiosis phases Several of the microscope activities that were identified as errors throughout the experiment will be thoroughly reviewed and rehearsed in the next practice sessions

REPORT 3: EXTRACTION OF DNA FROM

PLANT CELLS

1 Introduction:

An organism's genetic information is contained in its DNA, which is its genetic material In order to extract DNA from plant tissue, techniques like grinding with lysis buffers are used to break down the cell walls and membranes To purify DNA, cell debris is precipitated by centrifugation, and DNA is precipitated from the aqueous phase and thoroughly washed to remove contaminating salts The purified DNA is then stored in sterile distilled water or suitable buffer This method reliably yields intact genomic DNA from plant tissue

- Isopropanol (ice cold)

- 70% Ethanol (ice cold)

- Mortar and Pestle

Step 1: Grind 500 mg of plant tissue to a fine paste in approximately 1500µl lysis buffer Step 2: Label 2 eppendorf tubes as 2 samples: 1 without RNase and 1 with RNase(will

be added at the end) Perform the following steps for both samples

Step 3: Transfer 500µl lysis buffer/plant extract mixture into each eppendorf Add 2µl proteinase K to each tube

Step 4: Incubate the mixture for about 15 mins at 55°C in a recirculating water bath Step 5: After incubation, spin the mixture at 9000 rpm at 4°C for 10 mins to spin down cell debris Transfer 300µl supernatant to a clean eppendorf

Step 6: Add 300µl of Chloroform: IsoAmyl Alcohol (24:1) to the supernatant and mix the solution by inversion

Step 7: After mixing, spin the tubes at 9000 rpm at 4°C for 2 mins

Step 8: Transfer the upper aqueous phase only (contains the DNA) to a clean eppendorf Step 9: To each eppendorf add 0.1 volume of sodium acetate 3M pH 5.2 and 2 volumes

of ice cold isopropanol

Step 10: Invert the tubes slowly for a few times to precipitate the DNA Generally the DNA can be seen to precipitate out of solution

Step 11: Incubate the tube at -20°C for 15 mins to precipitate the DNA

Step 12: Spinning the tube at 9000 rpm at 4°C for 5 mins to form a pellet

Step 13: Remove the supernatant and wash the DNA pellet by adding 400ul ice cold 70% ethanol and spinning the tube at 9000 rpm for 5 minutes

Step 14: Remove the supernatant Wash the precipitate again with 400ul ice cold 70% ethanol

Step 15: After the wash, Remove all the supernatant and allow the DNA pellet to air dry (approximately 15 min) Do not allow the DNA to over dry or it will be hard to re-dissolve

Step 16: Resuspend the DNA in 30-40 ul sterile DNase free water

- Add 1ul RNase (10 µg/ml) to the water prior to resuspending the DNA in the “with RNAse” labeled tube

-After resuspension, the DNA is incubated at 65°C for 20 min to destroy any Dnases that may be present and stored at 4°C

3 Results:

Both samples (with and without RNAse) show very little to no DNA precipitation This might

be due to several factors which will be discussed further in the next report

4 Discussion:

Mechanism of the extraction:

- Lysis Buffer: Contains buffering salts, ionic salts to regulate the pH and osmolarity of the lysate and detergent to break up membrane structures

- Proteinase K: A serine protease that hydrolyzes peptide bonds, thus degrading proteins and eliminating contaminating RNase and Dnase The proteinase K has the highest activity in the range between 37℃ and 70℃, so the sample should be incubated in 55℃ after adding it to ensure complete digestion

- Chloroform-isoamyl: Chloroform-isoamyl contains chloroform and isoamyl alcohol When

it is added into the supernatant and centrifuged, Chloroform-isoamyl promotes the

partitioning of the solution into phases: The aqueous phase with DNA is at the top; the

organic hydrophobic phase with lipid, proteins, and other impurities is settled on the bottom; and the precipitated protein is denatured and coagulated between both these phases

- Sodium acetate and isopropanol: Sodium acetate is dissociated to CH3COO- and Na+, a cation which neutralizes by binding to negative charge points of the DNA, makes the complex less soluble and forms white precipitate On the other hand, isopropanol helps shield the Na+ PO3- complex from water molecules and pull it inside to precipitate

- The purpose of centrifugation: separate out two materials with different densities, such as DNA-containing solution and cell debris, solid phase DNA out of the solution

5 Conclusion:

After the session, students had the opportunity to use micropipette and learn about sample preparation and the procedures of extracting DNA from plant cells

REFERENCES:

[1] Chauhan, T (2023, October 24) Phenol chloroform DNA extraction: basics, preparation

of chemicals and protocol Genetic Education

REPORT 4: DNA QUALITY AND

QUANTIFICATION

1 Introduction:

The common laboratory technique for sorting DNA according to size (such as base pair length) for purification and visualization is gel electrophoresis Using an agarose gel matrix and an electrical field, electrophoresis transfers negatively charged DNA toward a positive electrode Shorter DNA fragments can travel across the gel matrix faster than bigger ones As

a result, a DNA ladder—a group of known-length DNA fragments—and an agarose gel can

be used to precisely measure the length of a DNA segment

2 Procedure:

Materials:

- Electrophoresis chamber & power supply

- Sample combs

- Electrophoresis buffer: SB buffer

- Loading dye, which contains something dense (e.g glycerol) to allow the sample to

"fall" into the sample wells

1 Place the gel plate into gel mould, position the comb

2 Prepare 0.8% agarose gel: dissolve 0.4 grams of agarose and add to the 50ml 1x SB buffer solution Heat the mixture in a microwave oven until completely dissolved Let the solution cool at room temperature to 60°C

3 Pour the agarose into a gel tray with the well comb in place, and allow it to set for at least 30 minutes Pour slowly to avoid bubbles which will disrupt the gel Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip

4 Remove the comb Place the gel into the gel electrophoresis chamber and fill the gel box with a 1x SB buffer until the gel is covered

5 Dye the DNA sample: Pipette 2 μl dye as well as 10 μl sample and mix them

together in waxed paper, then, adjust the pipette to 6 μl and take all to solution to the well, repeat the step to the other sample