Molecular Genetics LSM1102

Practical 1- 4 Instructions

Wu Jinlu dbswjl@nus.edu.sg

Tel: 65168476    

SAFETY REQUIREMENTS Lab coats must be worn at all times in the lab, but removed before leaving the laboratory.

Goggles must be worn when handling dangerous chemicals.

Suitable footwear must be worn to protect against spills; do not wear sandals or slippers.

Long hair should be tied back.

Do not eat, drink or apply cosmetics in the lab (especially nail polish – flammable

solvents).

Never use an open flame in the vicinity of flammable solvents.

Never leave a lighted burner unattended, keep a lighted Bursen burner an enough distance

from the shelf.

If you have an abrasion or cut on your hands, cover it before you begin work.

Report any accident to the demonstrators or instructors immediately.

Never pipette anything by mouth.

Wash your hands thoroughly with soap and water before leaving the lab.

Familiarize yourself with any additional rules relating to the laboratory in which you are

working. Note the location of eye wash points, safety shower, fire extinguisher and first

aid cabinets.

CONTENT

Practical 1 Session 1: Extraction of Genomic DNA from E. coli Session 2: Basic Bacterial Genetics Manipulations Practical 2 Session 1: Extraction of Plasmid DNA from E. coli Session 2: Basic Bacterial Genetics Manipulations (continued) Practical 3 Session 1: Transformation Session 2: Basic Bacterial Genetics Manipulations (continued) Practical 4 Session 1: Observation of results of the transformation Session 2: Basic Bacterial Genetics Manipulations (continued) Laboratory Venue: LS Lab 9 @MD4 Level 4 Time: 14:00-18:00 hours Attendance Please sign on the attendance sheet with your TA. Those who did not attend the lab session will be subject to a 50% penalty of their final report score.

Please arrive on time for your practical classes. Due to safety concerns, if you miss the pre-laboratory briefing, you will not be able to perform the experiment for that day.

Lab report (5 marks)

You are asked to write ONE lab report based on the following guidelines and lab content.

Report title Please use the title below for your report and submission. It will facilitate our marking and recording. • Matriculation No_Plasmid DNA Purification and Transformation. For example: U074274W_Plasmid DNA Purification and Transformation

Introduction (6%)

• Background and objective of the practical. Materials and Methods (4%)

• Briefly describe the materials and methods in less than 50 words. Please do not copy the details from the instruction booklet.

Results (45%)

• The data is organized in a clear manner.

• Figures and/or tables are effective and accompanied by titles and legends.

• Describe your results in text. Discussion (35%) (Interprets the results and reaches a conclusion)

• Explanation of basic principles

• Explanation of the experimental logic

• Explanation of nay difference between your results with others, or with your expectations.

Writing (10%)

• Text is clear, concise and easy to read. Submission

• Please upload your report to the “Lab report” folder of the “Submission of Assignments” Workbin by due time. We do not accept a hard copy of your report.

• To ensure that the correct file was successfully uploaded to the Workbin folder, please login to the IVLE website again a few minutes after you have logged-out. Download your file to verify that it is the correct file.

• It is your responsibility to ensure that the correct file was uploaded successfully to the Workbin folder. You will not be given a second chance to upload another file to the Workbin folder after the deadline has passed.

• If you decide to upload an updated version of your submission before the deadline, please delete the older file before uploading the newer file.

• There will be a 50% penalty for documents uploaded to the “lab report (Late Submission)” folder.

• There will be no marks for those who missed the late submissions deadline

• Your report will be examined by Turnitin program for plagiarism prevention (http://www.cit.nus.edu.sg/plagiarism/).

LSM 1102 PRACTICAL 1 I - Extraction of genomic DNA from E. coli Materials

1. E. coli pellet (the strain used for this experiment does not contain any plasmid) 2. 5 ml lysis buffer (50 mM EDTA, 0.1M NaCl, pH 7.5) 3. Lysozyme solution (20 mg/ml) 4. 2% SDS (sodium dodecyl sulphate)/2N NaOH solution 5. Phenol – equilibrated in TE 6. 3M Sodium Acetate solution (pH 5.2) 7. Ice-cold Ethanol 8. Ethanol, 70% 9. Water

Procedure

1. Resuspend E. coli pellet in 5 ml lysis buffer. 2. Add 0.2 ml lysozyme solution to the bacterial suspension. Mix by vortexing and

incubate for 10 min at 37°C. 3. Add 0.5 ml 2% SDS/2N NaOH solution. Mix gently and incubate for 5 min at

37°C or longer till the cell suspension becomes translucent. 4. Add 5 ml equilibrated phenol (Do this step in fume hood). Tightly close the

tube, mix gently by inverting the tube back and forth for 10 min. 5. Centrifuge at 3,000 rpm for 15 min. Transfer 2.5 ml of the upper aqueous phase

into a clean plastic tube. 6. Add 0.1 volume (i.e. 0.25 ml) of sodium acetate and mix gently. 7. Add 2 volumes (i.e. 5 ml) of ice cold ethanol, let it stand for 1 min and then

gently invert the tube. 8. Observe the strands of DNA precipitate being formed. 9. Pull up the strands of DNA with a glass rod. 10. Rinse DNA with 70% EtOH, transfer DNA into new eppendorf tube. 11. Add 200 μl water to DNA. This DNA solution will be used in the next practical.

II - Basic Bacterial Genetics Manipulations This experiment will continue to practical 4, in a total of 4 sessions with different experimental purpose. In this practical, two E. coli strains with different genetic materials are examined for their behaviours on different indicator plates.One strain (DH5α) is sensitive to ampicillin and incapable of utilizing lactose, i.e. lac -. The other strain (DH5α carrying pUC18) is resistant to ampicillin, and able to utilize lactose. These two strains will be streaked onto LB agar, LB + ampicillin, LB + Xgal + IPTG and MacConkey agar plates for observing their growth and appearance. In order to ensure purity of cultures, single colonies must be obtained. Apart from the technique of streaking for single colonies, replicaplating is also often used in bacterial genetics for the rapid screening of many strains for the desired phenotype. In this practical, E. coli colonies will be screened by replicating on the four different plates. Materials

1. Overnight broth cultures of E. coli DH5α and DH5α (pUC18) 2. Luria-Bertani (LB; tryptone-10g/l, yeast extract-5g/l, NaCl-5g/l, glucose-1g/l, agar1.5g/l) 3. LB plate – 2 4. LB + ampicillin – 2 5. LB + Xgal + IPTG – 2 6. MacConkey agar plates – 2

Objective Obtaining single isolated colonies Procedure

1. Streak each strain with a sterile wire loop to obtain single colonies on LB, LB + Ampicillin, LB + Xgal + IPTG and MacConkey plates. Streaking is a basic technique for obtaining a single clone, please ask the demonstrator to demonstrate if you donot know how to do.

2. Bring your plates to the indicated bench, otherwise, they will be trashed. 3. The lab staff will place your plates in incubator (37°C) for bacteria growth.

Effective Learning_practical I

Group No._________________; Matriculation No.____________________

Answer the following questions with True/False after the experiment

Each E. coli cell contains one copy of circular dsDNA (haploid).

All E. coli strains are harmful pathogens. We should handle them carefully.

We need to break up both cell and nuclear membrane to release the E. coli genome DNA.

Phenol at pH8.0 denatures proteins but does not denature DNA

DNA is not soluble in phenol.

EDTA chelates metal ions which may be needed for the activity of nuclease

Washing DNA with 70% ethanol is to remove the salt and DNase residues.

Lysozyme is a hydrolase that acts to break covalent bonds within the carbohydrate portion of the peptidoglycan wall that surrounds gram negative bacteria like E. coli.

A single molecule of genomic DNA of E. coli is so large that I can see with my naked eyes.

All the four types of plates used in this practical can be used for screening.

Q1. What is the function of sodium acetate used for genome DNA isolation?

Q2. What impressed you most in this practical? A:

Q3. What’s your suggestion to TA or instructor’s briefing

Your Question (s)

LSM 1102 PRACTICAL 2 I - Extraction of plasmid DNA from E. coli Introduction The High-Speed Plasmid Mini Kit is designed for rapid isolation of plasmid or cosmid DNA from 1-4 ml bacterial cultures. Modified alkaline Lysis method (1) and RNase treatment are used for obtaining clear cell lysate with minimal genomic DNA and RNA contaminants. In the presence of a chaotropic salt, the plasmid DNA in the lysate binds to the glass fiber matrix in the spin column (2). The contaminants are washed off with an ethanol contained Wash Buffer and the purified plasmid DNA is eluted by a low salt Elution Buffer or water. The procedure does not require DNA phenol extraction or alcohol precipitation. Typical yields are 20-30 μg for high-copy number plasmid or 3-10 μg for low-copy number plasmid. The entire procedure can be completed within 30 minutes. The purified plasmid DNA is ready to use for restriction enzyme digestion, ligation, PCR, and sequencing reactions. Materials

1. E. coli (pUC18) culture 2. Geneaid Spin Column with glass fiber matrix 3. Microcentrifuge tubes 4. Buffer PD1(50 mM Tris-HCl pH 8.0; 10 mM EDTA; 10 μg/ml RnaseA) 5. Buffer PD2 (200 mM NaOH; 1% SDS (w/v)) 6. Buffer PD3 (guanidinium chloride and acetic acid) 7. W1Buffer (guanidine hydrochloride and isopropanol) 8. Wash Buffer (70% ethanol) 9. Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)

Procedure

• Add provided RNase A to PD1 Buffer and store at 4ºC, if precipitates have formed in PD2 Buffer, warm the buffer in a 37ºC water bath to dissolve.

• Add absolute ethanol to Wash Buffer prior to initial use (see the bottle label for volume).

1. Harvesting E. coli cells. Transfer 1.5 ml of bacterial culture to a 1.5 ml microcentrifuge tube. Spin for 1

minute in a microcentrifuge and discard the supernatant. If more than 1.5 ml of bacterial culture is used, repeat the Harvesting Step.

2. Re-suspension

Add 200 μl of PD1 Buffer (RNase A added) to the tube and resuspend the cell pellet by vortex or pipetting.

3. Lysis Add 200 μl of PD2 Buffer and mix gently by inverting the tube 10 times. Do

not vortex to avoid shearing the genomic DNA. Let stand at room temperature for 2 minutes or until the lysate is homologous.

4. Neutralization Add 300 μl of PD3 Buffer and mix immediately by inverting the tube 10 times.

Do not vortex. Microcentrifuge for 3 minutes.

5. DNA Binding Place a PD Column in a 2 ml Collection Tube. Add the supernatant from Step 4 to the PD Column and microcentrifuge for

30 seconds. Discard the flow-through and place the PD Column back in the 2 ml Collection

Tube. 6. Wash

Add 400 μl of W1 Buffer into the center of the PD Column. Microcentrifuge for 30 seconds. Discard the flow-through and place the PD Column back in the 2 ml Collection

Tube. Add 600 μl of Wash Buffer (ethanol added) into the center of the PD Column. Microcentrifuge for 30 seconds. Discard the flow through and place the PD Column back in the 2 ml Collection

Tube. Microcentrifuge again for 3 minutes to dry the column matrix.

7. DNA Elution Transfer the dried PD Column to a new 1.5 ml microcentrifuge tube. Add 50 μl of Elution Buffer or water into the center of the column matrix. Let stand for 2 minutes or until the Elution Buffer or water is absorbed by the

matrix. Microcentrifuge for 2 minutes to elute the DNA.

References

(1) Birnboim, H. C. and Doly, J. (1979) Nucleic Acids Res. 7, 1513. (2) Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615.

II – Agarose gel electrophoresis of DNA Materials

1. DNA from Practical 1 2. Agarose 3. Ethidium bromide, 10 mg/ml, (**CARCINOGENIC**) 4. Tris-acetate buffer (TAE; 4mM Tris-acetate, 0.1mM EDTA) 5. Gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll 400) in water 6. Lambda HindIII-cleaved DNA marker 7. Water

Procedure

1. Prepare a 0.7% agarose solution (i.e. 0.7 g agarose in 100 ml TAE buffer) and heat to melt the agarose. Allow the solution to cool slightly and add 5 μl of ethidium bromide (Prepared by laboratory staff).

2. Pour into casting tray and allow agarose to set. Gels can be cast in the cold room in order to reduce setting time (Prepared by laboratory staff).

3. Prepare DNA samples by adding 2 μl Gel loading buffer to 18 μl DNA. 4. Place the gel and its casting tray into the gel tank; pour in sufficient TAE buffer to

just submerge the gel. Load DNA samples. (Note the size of sample wells and do not allow DNA to spill into the adjacent wells).

5. Run at 10-15 V/cm for at least 30 min. 6. Observe DNA bands on a UV transilluminator.

III - Basic Bacterial Genetics Manipulations (continued) Materials

LB agar plates – 2

Choose single colonies

• Examine the plates on which you streaked for single colonies.

• Using the grids provided, place an agar plate over the grid and pick 20 well isolated colonies from the various agar cultures onto each of the gridded positions.

(Making sure that you have recorded which plates the colonies came from and their appearance on the plates.)

• Bring your plates to the indicated bench.

• Incubate the plates for growth.

Effective Learning_Practical II

Group No._____________; Matriculation No.__________________

Answer the following questions with True/False

Plasmid DNA is one kind of genetic material which can pass to the next generation during E. coli replication.

All plasmid DNAs carry ampicillin resistant gene.

Bacterial chromosomal DNA attaches to cell membrane, so it can be co-precipitated with insoluble complexes during plasmid purification.

DNA size is the only factor which affects its migration speed during agarose gel electrophoresis.

One E.coli cell contains one copy of plasmid and one copy of chromosomal DNA

Ethidium bromide is a strong mutagen and a possible carcinogen

The DNAs are negatively charged but proteins are positively charged.

DNA loading buffer allows the DNA be negatively charged so it can move toward anode during electrophoresis.

1% agarose gel is prepared by melting 1 gram agarose into 100 ml distilled water.

Do you know the method(s) how to assess your DNA purity and quantity?

Q1. What is the function of buffer PD3?

Q2. What impressed you most in this practical? A:

Q3. What’s your suggestion to TA or instructor’s briefing?

Your Question (s)

LSM 1102 PRACTICAL 3 I - Transformation Materials

1. Competent E. coli cells 1 ml 2. Plasmid DNA (pUC18) _prepared in practical 2 3. Double distilled (dd) sterile water 4. LB broth 5. LB agar plates _(one plate for each pair) 6. LB + ampicillin agar plates_(5 plates for each pair)

Procedure

1. Set up 3 microfuge tubes with 300 μl competent cells in each tube. 2. Add 5 μl and 15 μl plasmid DNA to 2 tubes and 10 μl dd water to the third. 3. Leave on ice for 30 min. 4. Heat shock at 42°C for 90 seconds. 5. Add 1 ml LB broth and incubate at 37°C for 40 min. Invert the tube at 10 min

intervals to mix. 6. Centrifuge at 12,000 rpm for 2 min. Discard all supernatant. 7. Re-suspend in 300 μl LB broth. Make a 10-1

dilution of only the transformed cells. 8. Spread 100 μl of the neat and 10-1

suspensions on LB and LB + ampicillin agar plates. (100 ul neat of negative control to each of LB and LB+ampicillin plates)

9. Incubate overnight at 37°C.

II - Basic Bacterial Genetics Manipulations (continued) Materials

1. LB agar plate – 2 plates for each pair of students 2. LB + ampicillin agar plates– 2 3. LB + Xgal + IPTG agar plates - 2 4. MacConkey agar plates – 2

Procedure

Transfer the bacteria of gridded plate (master plate) onto a velvet held firmly on a block by a metal ring and then replicating from the velvet onto an LB; LB + ampicillin; LB + IPTG + Xgal and MacConkey agar plates. Bring your plates to the preparation bench (between B2 and B3)! Incubate the plates for growth.

Effective Learning_Practical III

Group No._____________; Matriculation No.__________________

Answer the following questions with True/False

Bacteria in different phase of growth are equally competent for taking foreign DNA.

Calcium ions improve the transformation efficiency by neutralizing the negative charges on DNA molecules and phospholipids.

The ampicillin-resistance gene codes for the enzyme β-lactamase that destroys ampicillin. The enzyme is secreted into the cell’s environment, so that the antibiotic is destroyed before it even enters the cell.

The number of cells transformed per 1 microgram of DNA is called the transformation efficiency.

In the plasmid transformation, after the heat shock step intact plasmid DNA molecules can replicate in bacterial host cells.

Transferred gene must integrate into a host cellular chromosome to be transcribed and translated.

Linear plasmid DNA gives higher transformation efficiencies than supercoiled plasmid DNA.

Human/animal cells can also be transformed with a foreign DNA.

If Mammalian cells take up the plasmid pUC18, the cells can also produce β-lactamase to hydrolyze ampicillin.

Transgenic animals can express foreign proteins because their cells contain the plasmid carrying the gene.

Q1. How are bacterial cells containing plasmid DNA selected?

Q2. What impressed you most in this practical? A:

Q3. What’s your suggestion to TA or instructor’s briefing?

Your Question (s)

LSM 1102 PRACTICAL 4

I - Basic Bacterial Genetics Manipulations (continued)

Examine the replicated plates.

Some grids on some of the plates will not show any growth as some of the

colonies on the original LB plate culture are not resistant to the antibiotics but all

the grids on LB plate should show growth. Some strains are also unable to use

lactose or X-gal.

II - Transformation (continued)

Examine the agar plates. Note the relative number of transformants on the

different plates.