LMBTY312: Lab in Genetic Engineering

Laboratory Manual BTY312 Genetic Engineering

LMBTY312: Lab in Genetic Engineering

Table of Contents S. No. 1 2 3 4 5 6 7 8 Title of the Experiment DNA isolation from plants DNA isolation from E. coli Spectrophotometer analysis of DNA Agarose Gel Electrophoresis of DNA Plasmid DNA isolation Restriction digestion of DNA Bioinformatics tool: Restriction enzymes Rebase, Neb Cutter 3 4 6 6 7 8 for 10 10 Page no.

Bioinformatics tool: Primer design

TEXT BOOK(S): nd 1. J. Sambrook , E.F. Fritsch and T. Maniatis (1989) Molecular Cloning A laboratory Manual 2 ed. , Cold Spring Harbor Laboratory press

LMBTY312: Lab in Genetic Engineering

1.Experiment no. 1: DNA isolation from plants Equipment Required: Centrifuge, Pestle and Mortar Material Required:
2X CTAB Extraction Buffer (pH 8.0) [ 2% CTAB, 100 mM Tris HCl, 20 mM EDTA, 1.4 M NaCl, 2l /ml Mercaptoethanol ( add just before used )], Chloroform / Isoamyl alcohol [Mix
24 volume of chloroform with 1 volume of isoamyl alcohol], 70% Ethanol, Isopropanol, T. E. Buffer (pH 8.0).

2. Learning Objectives:
To learn the rationale and mechanism of DNA isolation from plant leaves.

3. Outline of the Procedure:

Grind 1gm of leaves in a mortar and pestle (Mid-ribs of the leaves could be eliminated). Add 5ml of pre-warmed (at 65oC) 2X CTAB DNA extraction buffer per gram of tissue and grind a little more. Transfer this homogenized solution in a sterilized centrifuge tube. Incubate at 65oC for 1 hr. in a water bath. Add 0.6 Vol of chloroform-isoamyl alcohol (24:1). Mix by inversion for 15 min. Spin at 15,000rpm for 15 min. Three layers become visible; the upper aqueous layer contains DNA, the middle layer contains precipitated proteins, and the bottom layer contains cell debris and chloroform. Transfer the upper aqueous phase to another tube and estimate its volume. Add twice the volume of absolute alcohol or 0.6 Vol of isopropyl alcohol to precipitate the DNA. Centrifuge at 10,000rpm for 5min. to pellet the DNA. Discard the supernatant carefully without disturbing the pellet. Wash the pellet with 1ml of 70% ethyl alcohol. Incubate it at -200C for 15 min. and centrifuged at 15,000rpm for 5 min. Discard the supernatant carefully without disturbing the pellet. Air-dry the pellet till all the ethanol evaporates. Dissolve the dried DNA pellet in 200l TE buffer (pH8.0).

4. Scope of the results expected: The student will be able to isolate DNA and visualize it
in gel electrophoresis.

5. Parameters and Plots: None 6. Cautions:

handle gels.
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For visualizing DNA be careful while using UV lamp. Always use gloves to

LMBTY312: Lab in Genetic Engineering

1.Experiment no. 2: DNA isolation from E. coli Equipment Required:

pH meter, weighing balance, centrifuge.

Material Required:
Microbial culture, T.E. Buffer (pH 8.0), 5 M NaCl, CTAB/NaCl (Dissolve 4.1g NaCl and 10g CTAB in 100ml distilled water at 65oC and store at room temperature.), Chloroform / Isoamyl alcohol (24:1), 10% SDS, Lysozyme (Dissolve 20 mg lysozyme in 1ml deionized distilled water. Store the solut on n small al quots at -2 C), Proteinase K (Dissolve 10mg of proteinase -K in 1 ml deionized distilled water and store the solution at -2 C), 70% Ethanol, Isopropanol.

2. Learning Objectives:
To learn the rationale and mechanism of DNA isolation from bacterial cells.

3. Outline of the Procedure:

Scrap 1 or 2 loop full of microbial growth from culture media and suspend it in 400l of T.E. buffer in a vial.

Freeze and thaw it by keeping the vial at -200C for 15 min. heat it immediately up to 800C-1000C for 5 min. and again snap cool it by keeping the vial in ice for 15 min. Repeat the step.

Add 40l lysozyme, mix well & incubate for 2 hrs at 370C in shaking water bath.

Add 56 l of 10% SDS and 5l of proteinase K; mix well & incubate at 650C in shaking water bath for 30min.

Add 80l of 5M NaCl and 64l of CTAB/ NaCl solution and incubate at 650C in water bath for 30 min.

LMBTY312: Lab in Genetic Engineering

Add equal volume of freshly prepared chloroform/Isoamyl alcohol solution (24:1) mix well & centrifuge at 8000 x g for 15min. Three layers become visible; the upper aqueous layer contains DNA which is taken into another fresh microcentrifuge tube.

Add 0.6 volume of isopropanol to the supernatant and incubate at -200C for 30 min.

Centrifuge the tube at 8000 x g for 5 min.

Discard the supernatant without disturbing the pellet.

Add 150l of 70% chilled ethanol; centrifuge the tube at 8000 x g for 5 min.

Discard the supernatant and air dry the pellet.

Add 30l T.E. Buffer and store at -200C.

4. Scope of the results expected: The student will be able to isolate DNA and visualize it
in gel electrophoresis.

5. Parameters and Plots: None 6. Cautions:

handle gels. For visualizing DNA be careful while using UV lamp. Always use gloves to

LMBTY312: Lab in Genetic Engineering

1.Experiment no. 3: Spectrophotometer analysis of DNA Equipment Required:

UV-VIS spectrophotometer.

Material Required:
DNA solution, TE buffer.

2. Learning Objectives:
To quantify the amount of DNA in the solution.

3. Outline of the Procedure:

1. Take 2ml TE buffer in cuvette and calibrate spectrophotometer at 260nm. 2. Now prepare samples of known concentration of DNA in 4 eppendorf tubes.

3. Measure the absorbance of these sample and calculate the amount of DNA. 4. Scope of the results expected: The student will be able to calculate the amount of DNA
present in the sample.

5. Parameters and Plots: Concentration versus absorbance. 6. Cautions:

Cuvette used for measuring OD should be clean, wait for measuring the OD until the value stabilizes.

1.Experiment no. 4: Agarose Gel electrophoresis of DNA Equipment Required: Horizontal Gel Electrophoresis apparatus. Material Required: Tris Acetate EDTA Buffer (TAE Buffer) [50X- Tris base 242 g, Glacial
Acetic acid 37.1 ml, EDTA 37.2 g, adjust final volume up to 1000 ml with deionised distilled water, maintain pH to 8.0], Ethidium bromide dye [Ethidium bromide - 10mg, Distilled water-1 ml], Agarose, DNA loading dye [Bromo phenol Blue: 0.25%, Xylene cynol : 0.25%, Glycerol : 30%].

2. Learning Objectives:
The student will be able to detect whether the DNA has been successfully isolated or not.

3. Outline of the Procedure:

Dissolve agarose in 1X TAE buffer. 6

LMBTY312: Lab in Genetic Engineering

Gradually bring the solution to boil in a water bath mixing occasionally by swirling with hands. Cool the solution up to 550C and add ethidium bromide (0.5g/ml) to the solution and dispense the solution in electrophoretic unit with appropriate well forming comb. Pour TAE buffer in electrophoresis unit. Prepare gel in such a way that the wells are towards cathode. Load the sample in wells and run the gel at 50 V for 2 hrs. Observe the gel on U.V. Transilluminator.

4. Scope of the results expected: The student will be able to isolate DNA and visualize it
in gel electrophoresis.

5. Parameters and Plots: None 6. Cautions: Ethidium Bromide is a powerful mutagen and is toxic. Avoid breathing the dust.
Wear gloves when working with solutions that contain EtBr. Be careful while plugging the wires of cathode and anode of electrophoresis unit.

1. Experiment no. 5: Plasmid DNA isolation Equipment Required: Centrifuge, weighing balance. Material Required: LB (Luria-Bertani) Broth, GTE solution [50mM glucose,10mM EDTA,
25mM Tris-HCl], NaOH/SDS Lysis Solution [0.2N NaOH, 1% SDS], 5M Potassium Acetate Solution [5 M potassium acetate 60 ml, glacial acetic acid 11.5 ml, ddH2O 28.5 ml], TE Buffer [10 mM Tris-Cl, pH 7.5, 1 mM EDTA], RNAse A solution (20 mg/ml).

2. Learning Objectives:
Plasmid is a vector being routinely used in genetic engineering. The experiment enables students to be able to isolate plasmid from bacterial cell.

3. Outline of the Procedure:

1. Pick single colony and inoculate 5 ml of LB broth containing 200 g/l ampicillin. 2. Centrifuge 1.5ml broth at 10,000rpm for 5 minute. Aspirate & discard supernatant. 3. Resuspend cell pellet in 100 l of GTE buffer 4. Add 200 l of NaOH/SDS lysis solution. Invert tube 6-8 times. 7

LMBTY312: Lab in Genetic Engineering

5. Immediately add 150 l of 5 M potassium acetate solution (pH 4.8). This solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble white, rubbery precipitate. Spin at 15,000rpm for 10min. 6. Transfer supernatant to new tube, being careful not to pick up any white flakes. Precipitate the nucleic acids with 0.5mL of isopropanol on ice for 10-20 minutes and centrifuge at 15,000rpm for 5 minute. 7. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 0.4 ml of TE buffer. Add 10l of RNAse A solution (20 mg/ml stock stored at -20 C), vortex and incubate at 37 C for 20 to 30 minutes to digest remaining RNA. 8. Extract proteins from the plasmid DNA using PCIA (phenol/chloroform/isoamyl alcohol in 25:24:1) by adding about 0.3 ml. Vortex vigorously for 30 seconds. Centrifuge at 15,000rpm for 5 minutes at room temperature. 9. Remove upper aqueous layer containing the plasmid DNA carefully avoiding the white precipitated protein layer above the PCIA layer, transferring to a clean 1.5 ml epindorf tube. 10. Add 100 l of 7.5 M ammonium acetate solution and 1 ml of absolute ethanol to precipitate the plasmid DNA on ice for 10 minutes. Centrifuge at 15,000rpm for 5 minutes at room temperature.

11. Aspirate off ethanol solution, air-dry and resuspend DNA pellet in 50l of TE Buffer.
.

4. Scope of the results expected: The student will be able to isolate plasmid. 5. Parameters and Plots: None 6. Cautions:
Acetic acid (concentrated) must be handled with great care. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and goggles. SDS (Sodium dodecyl sulfate) is toxic, an irritant, and poses a risk of severe damage to the eyes. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

1.Experiment no. 6: Restriction digestion of DNA Equipment Required: Horizontal electrophoresis kit. Material Required:
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LMBTY312: Lab in Genetic Engineering

Buffer Low Medium High Enzyme EcoRI PstI TaqI

NaCl 0 mM 50 mM 100 mM Buffer high med low

Tris 10 mM (pH7.4) 10 mM (pH7.4) 50 mM (pH7.4)

MgSO4 10 mM 10 mM 10 mM

Dithiothreitol 1 mM 1 mM 0 mM

Incubation Temperature 37oC 21-37oC 65oC

Recognition sequence G*AATTC CTGCA*G T*CGA

DNA sample (0.2-1.0g), Distilled Water.

2. Learning Objectives:
To do the restriction digestion of DNA and analyze the bands in the gel electrophoresis.

3. Outline of the Procedure:

1. The total volume of the reaction should be kept at 20 l. This is the amount typically run on a gel. The reaction mix may contain 0.2 - 1.0 g DNA. The amount of DNA added depends on the DNA prep. The volume of DNA may vary from 5l to 10l. 2. Add 2 l of 10x restriction buffer (for final conc. up to 1X). 3. Add 2l-5l of restriction enzyme (5U-10U for plasmid or prokaryoitic DNA & 20U-30U for eukaryotic DNA). 4. Add sterile distilled water to make the final volume up to 20l. Tap the tube several times to ensure mixing. 5. Centrifuge the tubes briefly (turn centrifuge on, allow it to get to speed, and turn it off) to concentrate all of the liquid at the bottom. 6. Place in water bath for 30 - 60 minutes. Typically this is at 37oC. (Note: Incubation Temperature may vary according to the enzyme used.) Analyze the results on a gel, add 5 l of tracking dye to the sample and load the samples into a well on an agarose gel.

4. Scope of the results expected: The student will be able to analyze and perform
restriction digestion of DNA by visualizing DNA bands in agarose gel electrophoresis.

5. Parameters and Plots: None

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LMBTY312: Lab in Genetic Engineering

6. Cautions:

Restriction enzyme must be handled with care and should not be wasted. Be careful while plugging the wires of cathode and anode of electrophoresis unit. Wear gloves while working with agarose gels.

1.Experiment no. 7: Bioinformatics tool: Rebase, Neb Cutter for Restriction enzymes Equipment Required: Computer with internet connection. Material Required: None. 2. Learning Objectives:
To study the importance of various biological databases and information content and analysis possible with tools.

3. Outline of the Procedure:

Go to Rebase and Neb Cutter home page with help of any search engine. Get familiar with the data types and information available at the database.

4. Scope of the results expected: Analysis of recognition and restriction site of different
restriction enzymes.

5. Parameters and Plots: None 6. Cautions: None.

1. Experiment no. 8: Bioinformatics tool: Primer design Equipment Required: Computer with internet connection. Material Required: None. 2. Learning Objectives:
To study the importance of various biological tools and analysis possible with these tools. Primer designing is a step routinely followed to selectively amplify DNA fragment.

3. Outline of the Procedure:

Go to the home page of the tool with help of any search engine. Get familiar with the types of analysis possible with the tool. 10

LMBTY312: Lab in Genetic Engineering

4. Scope of the results expected: Analysis of approach to be followed to selectively

amplify DNA fragment.

5. Parameters and Plots: None 6. Cautions: None.

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