TABLE OF CONTENTS
S. No. Title of the Experiment Page No.
1 Extraction and Purification of Chromosomal DNA from E. coli 5
Extraction and Purification of Plasmid DNA from E. coli by
2 Alkaline Lysis Miniprep Method 10
Preparation of Insert DNA by Fragmentation and its Semi-
3 Quantitative Analysis 14
Preparation of Insert DNA by PCR amplification and its Semi-
4 Quantitative Analysis 18
5 Preparation of TA Cloning Vector 22
6 Purification of Insert and Vector DNA 26
7 Ligation of Insert and Vector DNA 30
Preparation of Chemically Competent E. coli DH5α and
8 Transformation with Recombinant DNA 34
Blue-White Screening through Alpha-Complementation and
9 Determination of Transformation Efficiency 38
10 Colony PCR for Confirmation of Gene Cloning 43
Reference Books:
1. Laboratory Manual for Genetic Engineering by Vennison S. John, Prentice Hall, 1st Edition
(2009).
LMBTY614 4
, Experiment No. 1
1. Experiment: Extraction and Purification of Chromosomal DNA from E. coli
Equipment Required: Water baths, UV Spectrophotometer, Centrifuge
Material Required: CTAB/NaCl solution: (10% CTAB in 0.7 M NaCl): Dissolve 4.1 g NaCl in
80 ml water and slowly add 10 g CTAB (hexadecyl trimethyl ammonium bromide) while heating
and stirring. If necessary, heat to 65 °C to dissolve. Adjust final volume to 100 ml. 10% sodium
dodecyl sulfate (SDS), 25:24:1 Phenol/chloroform/isoamyl alcohol, 1X TE buffer (pH 8.0): 10
mm Tris HCl (pH 8.0), 1 mm EDTA (pH 8.0), Isopropanol, Ethanol (ice cold), 70% Ethanol (ice
cold), Proteinase K, 5M NaCl, 50X TAE buffer (Tris base 242g/L, 0.5M EDTA 100mL/L, glacial
acetic acid 57.1mL/L)
2. Learning Objectives: Isolation of pure DNA from bacteria.
3. Theory: The isolation and purification of DNA from cells is one of the most common procedures
in contemporary molecular biology and embodies a transition from cell biology to the molecular
biology (from in vivo to in vitro). The isolation of DNA from bacteria is a relatively simple
process. The organism to be used should be grown in a favorable medium at an optimal
temperature (37°C), and should be harvested in late log to early stationary phase for maximum
yield. The genomic DNA isolation needs to separate total DNA from RNA, protein, lipid, etc.
Initially the cell membranes must be disrupted to release the DNA in the extraction buffer. SDS
(sodium dodecyl sulphate) is used to disrupt the cell membrane. Once cell is disrupted, the
endogenous nucleases tend to cause extensive hydrolysis. Nucleases apparently present on human
fingertips are notorious for causing spurious degradation of nucleic acids during purification.
DNA can be protected from endogenous nucleases by chelating Mg2+ ions using EDTA. Mg2+ ion
is considered as a necessary cofactor for action of most of the nucleases. Nucleoprotein
interactions are disrupted with SDS, phenol or proteinase K. Proteinase K enzyme is used to
degrade the proteins in the disrupted cell soup. Phenol and chloroform are used to denature and
separate proteins from DNA. Chloroform is also a protein denaturant, which stabilizes the rather
unstable boundary between an aqueous phase and pure phenol layer. The denatured proteins form
a layer at the interface between the aqueous and the organic phases which are removed by
centrifugation. DNA released from disrupted cells is precipitated by cold absolute ethanol or
isopropanol. Most commonly used protocols for the preparation of bacterial genomic DNA
consist of lysozyme/detergent lysis, followed by incubation with a nonspecific protease and a
series of phenol/chloroform/isoamyl alcohol extractions prior to alcohol precipitation of the
nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in
removing the copious amounts of exopolysaccharides that are produced by many bacterial genera,
LMBTY614 5
, and which can interfere with the activity of molecular biological enzymes such as restriction
endonucleases and ligases. In this procedure, however, the protease incubation is followed by a
CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein;
both groups of contaminating molecules are effectively removed in the subsequent emulsification
and extraction with chloroform/isoamyl alcohol. This procedure is effective in producing
digestible chromosomal DNA from a variety of gram-negative bacteria. DNA absorb strongly at
260 nm (max of DNA), proteins absorb strongly at 280 nm (max of proteins). Therefore, in
assessing the purity of the nucleic acid samples, we can use the ratio of measurements of these
wavelengths 260 nm, and 280 nm (the ratio should be between 1.65 and 1.85).
4. Outline of the Procedure:
i. Grow bacterial strain to saturation. Spin 2 ml culture for 2 min at 6000-8000 rpm in a micro
centrifuge.
ii. Re-suspend in 567μl TE buffer, 15μl of 10% SDS, 3.6μl of 20 mg/ml proteinase K. Mix and
incubate for 1 hr at 37°C. (The solution should become viscous as the detergent lyses the
bacterial cell walls. There should be no need to predigest the bacterial cell wall with
lysozyme).
iii. Add 100μl of 5 M NaCl. Mix thoroughly. (This step is very important since a CTAB–
nucleic acid precipitate will form if salt concentration drops below about 0.5 M at room
temperature. The aim here is to remove cell wall debris, denatured protein, and
polysaccharides complexed to CTAB, while retaining the nucleic acids in solution).
iv. Add 80μl of CTAB/NaCl (10% w/v; 0.7M) solution and mix. Incubate for 10 min at 65°C.
v. Centrifuge at 10,000 rpm for 10 minutes. Remove aqueous, viscous supernatant to a fresh
Falcon tube, leaving the interface behind. Add an equal volume of
phenol/chloroform/isoamyl alcohol, extract thoroughly and spin for 5 min. (This extraction
removes CTAB–protein/polysaccharide complexes).
vi. Transfer aqueous phase to a fresh tube. Add 1 volume of Isopropanol, mix and shake.
vii. Centrifuge at 10,000 rpm for 10 minutes. Remove the supernatant and add 1 ml 70% ethanol
to wash the pellet.
viii. Centrifuge the above contents at 10,000 rpm for 10 minutes. After air drying for 5 minutes,
dissolve the DNA in 200μl of TE buffer or double distilled H2O (ddH2O). (DNA should not
be over dried as re-suspension in TE become difficult).
ix. The concentration of DNA is determined using a spectrophotometer. DNA samples can be
stored at -20°C for further experiments.
x. If purification is required, take the supernatant at step 5 and add three volumes of chaotropic
salt solution (HiPura DNA purification kit) and follow the steps as per manufacturer’s
instructions. Elute the DNA in 30μL of TE buffer or sterile molecular biology grade water.
DNA Spectrophotometry
i. Prepare the DNA dilution by adding 10μl of the E. coli DNA stock in 2 ml of ddH2O in a
microcentrifuge tube and Mix it.
LMBTY614 6
, ii. Clean the quartz cuvette with ddH2O and set the spectrophotometer to read absorbance at 260
and 280 nm.
iii. Use a blank solution (TE buffer or ddH2O) to set up zero absorbance in the
spectrophotometer. To prepare a blank, add 2 ml of TE buffer or ddH2O to a clean cuvette.
Place the cuvette into the spectrophotometer, and press the “blank” button.
iv. Discard the ddH2O. Transfer the DNA sample into the cuvette and read the absorbance at 260
and 280 nm.
v. The concentration of pure double-stranded DNA with an A260 of 1.0 is 50μg/ml. Thus, one
can determine the DNA concentration of a solution.
Agarose Gel Electrophoresis
i. Dilute 50X TAE buffer 50-folds (1-part stock in 49-parts water) to make 1X TAE buffer.
ii. Weigh out 0.32 grams of agarose and add to 40mL of 1X TAE buffer and boil the suspension
till agarose dissolves completely.
iii. Make up the volume to 40 mL to account for volume loss by evaporation and add 5μl of
10mg/mL solution of ethidium bromide and mix well.
iv. Pour the gel into a sealed gel-casting tray and put the comb in place.
v. Allow the gel to solidify and set it up in the tank filled with 1X TAE buffer.
vi. Load 2μl of DNA onto the gel and run at 100V for 30 minutes along with a positive control
1μl lambda DNA or any other high-molecular weight DNA.
vii. Observe the gel in the gel documentation system and record the results.
4. Results: Qualitative and quantitative DNA analysis would tell about the amount and purity of the
DNA.
5. Scope of the result: DNA isolation depends on the accuracy during mixing of various reagents.
DNA pellet must be handled carefully.
6. Results Required: High quality and good quantity of DNA
7. Cautions:
a. Care should be taken to do the operations as gently as possible. Vortexing, pipetting
using fine tips etc. should be avoided to prevent the shearing of DNA.
b. Phenolic chloroform extraction step may be repeated if the aqueous layer is not clear
and transparent
8. Suggested Readings: Experiments in Molecular Biology: Biochemical Applications by Zachary
F. Burton, Academic Press Inc; Spl edition (2003).
LMBTY614 7
S. No. Title of the Experiment Page No.
1 Extraction and Purification of Chromosomal DNA from E. coli 5
Extraction and Purification of Plasmid DNA from E. coli by
2 Alkaline Lysis Miniprep Method 10
Preparation of Insert DNA by Fragmentation and its Semi-
3 Quantitative Analysis 14
Preparation of Insert DNA by PCR amplification and its Semi-
4 Quantitative Analysis 18
5 Preparation of TA Cloning Vector 22
6 Purification of Insert and Vector DNA 26
7 Ligation of Insert and Vector DNA 30
Preparation of Chemically Competent E. coli DH5α and
8 Transformation with Recombinant DNA 34
Blue-White Screening through Alpha-Complementation and
9 Determination of Transformation Efficiency 38
10 Colony PCR for Confirmation of Gene Cloning 43
Reference Books:
1. Laboratory Manual for Genetic Engineering by Vennison S. John, Prentice Hall, 1st Edition
(2009).
LMBTY614 4
, Experiment No. 1
1. Experiment: Extraction and Purification of Chromosomal DNA from E. coli
Equipment Required: Water baths, UV Spectrophotometer, Centrifuge
Material Required: CTAB/NaCl solution: (10% CTAB in 0.7 M NaCl): Dissolve 4.1 g NaCl in
80 ml water and slowly add 10 g CTAB (hexadecyl trimethyl ammonium bromide) while heating
and stirring. If necessary, heat to 65 °C to dissolve. Adjust final volume to 100 ml. 10% sodium
dodecyl sulfate (SDS), 25:24:1 Phenol/chloroform/isoamyl alcohol, 1X TE buffer (pH 8.0): 10
mm Tris HCl (pH 8.0), 1 mm EDTA (pH 8.0), Isopropanol, Ethanol (ice cold), 70% Ethanol (ice
cold), Proteinase K, 5M NaCl, 50X TAE buffer (Tris base 242g/L, 0.5M EDTA 100mL/L, glacial
acetic acid 57.1mL/L)
2. Learning Objectives: Isolation of pure DNA from bacteria.
3. Theory: The isolation and purification of DNA from cells is one of the most common procedures
in contemporary molecular biology and embodies a transition from cell biology to the molecular
biology (from in vivo to in vitro). The isolation of DNA from bacteria is a relatively simple
process. The organism to be used should be grown in a favorable medium at an optimal
temperature (37°C), and should be harvested in late log to early stationary phase for maximum
yield. The genomic DNA isolation needs to separate total DNA from RNA, protein, lipid, etc.
Initially the cell membranes must be disrupted to release the DNA in the extraction buffer. SDS
(sodium dodecyl sulphate) is used to disrupt the cell membrane. Once cell is disrupted, the
endogenous nucleases tend to cause extensive hydrolysis. Nucleases apparently present on human
fingertips are notorious for causing spurious degradation of nucleic acids during purification.
DNA can be protected from endogenous nucleases by chelating Mg2+ ions using EDTA. Mg2+ ion
is considered as a necessary cofactor for action of most of the nucleases. Nucleoprotein
interactions are disrupted with SDS, phenol or proteinase K. Proteinase K enzyme is used to
degrade the proteins in the disrupted cell soup. Phenol and chloroform are used to denature and
separate proteins from DNA. Chloroform is also a protein denaturant, which stabilizes the rather
unstable boundary between an aqueous phase and pure phenol layer. The denatured proteins form
a layer at the interface between the aqueous and the organic phases which are removed by
centrifugation. DNA released from disrupted cells is precipitated by cold absolute ethanol or
isopropanol. Most commonly used protocols for the preparation of bacterial genomic DNA
consist of lysozyme/detergent lysis, followed by incubation with a nonspecific protease and a
series of phenol/chloroform/isoamyl alcohol extractions prior to alcohol precipitation of the
nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in
removing the copious amounts of exopolysaccharides that are produced by many bacterial genera,
LMBTY614 5
, and which can interfere with the activity of molecular biological enzymes such as restriction
endonucleases and ligases. In this procedure, however, the protease incubation is followed by a
CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein;
both groups of contaminating molecules are effectively removed in the subsequent emulsification
and extraction with chloroform/isoamyl alcohol. This procedure is effective in producing
digestible chromosomal DNA from a variety of gram-negative bacteria. DNA absorb strongly at
260 nm (max of DNA), proteins absorb strongly at 280 nm (max of proteins). Therefore, in
assessing the purity of the nucleic acid samples, we can use the ratio of measurements of these
wavelengths 260 nm, and 280 nm (the ratio should be between 1.65 and 1.85).
4. Outline of the Procedure:
i. Grow bacterial strain to saturation. Spin 2 ml culture for 2 min at 6000-8000 rpm in a micro
centrifuge.
ii. Re-suspend in 567μl TE buffer, 15μl of 10% SDS, 3.6μl of 20 mg/ml proteinase K. Mix and
incubate for 1 hr at 37°C. (The solution should become viscous as the detergent lyses the
bacterial cell walls. There should be no need to predigest the bacterial cell wall with
lysozyme).
iii. Add 100μl of 5 M NaCl. Mix thoroughly. (This step is very important since a CTAB–
nucleic acid precipitate will form if salt concentration drops below about 0.5 M at room
temperature. The aim here is to remove cell wall debris, denatured protein, and
polysaccharides complexed to CTAB, while retaining the nucleic acids in solution).
iv. Add 80μl of CTAB/NaCl (10% w/v; 0.7M) solution and mix. Incubate for 10 min at 65°C.
v. Centrifuge at 10,000 rpm for 10 minutes. Remove aqueous, viscous supernatant to a fresh
Falcon tube, leaving the interface behind. Add an equal volume of
phenol/chloroform/isoamyl alcohol, extract thoroughly and spin for 5 min. (This extraction
removes CTAB–protein/polysaccharide complexes).
vi. Transfer aqueous phase to a fresh tube. Add 1 volume of Isopropanol, mix and shake.
vii. Centrifuge at 10,000 rpm for 10 minutes. Remove the supernatant and add 1 ml 70% ethanol
to wash the pellet.
viii. Centrifuge the above contents at 10,000 rpm for 10 minutes. After air drying for 5 minutes,
dissolve the DNA in 200μl of TE buffer or double distilled H2O (ddH2O). (DNA should not
be over dried as re-suspension in TE become difficult).
ix. The concentration of DNA is determined using a spectrophotometer. DNA samples can be
stored at -20°C for further experiments.
x. If purification is required, take the supernatant at step 5 and add three volumes of chaotropic
salt solution (HiPura DNA purification kit) and follow the steps as per manufacturer’s
instructions. Elute the DNA in 30μL of TE buffer or sterile molecular biology grade water.
DNA Spectrophotometry
i. Prepare the DNA dilution by adding 10μl of the E. coli DNA stock in 2 ml of ddH2O in a
microcentrifuge tube and Mix it.
LMBTY614 6
, ii. Clean the quartz cuvette with ddH2O and set the spectrophotometer to read absorbance at 260
and 280 nm.
iii. Use a blank solution (TE buffer or ddH2O) to set up zero absorbance in the
spectrophotometer. To prepare a blank, add 2 ml of TE buffer or ddH2O to a clean cuvette.
Place the cuvette into the spectrophotometer, and press the “blank” button.
iv. Discard the ddH2O. Transfer the DNA sample into the cuvette and read the absorbance at 260
and 280 nm.
v. The concentration of pure double-stranded DNA with an A260 of 1.0 is 50μg/ml. Thus, one
can determine the DNA concentration of a solution.
Agarose Gel Electrophoresis
i. Dilute 50X TAE buffer 50-folds (1-part stock in 49-parts water) to make 1X TAE buffer.
ii. Weigh out 0.32 grams of agarose and add to 40mL of 1X TAE buffer and boil the suspension
till agarose dissolves completely.
iii. Make up the volume to 40 mL to account for volume loss by evaporation and add 5μl of
10mg/mL solution of ethidium bromide and mix well.
iv. Pour the gel into a sealed gel-casting tray and put the comb in place.
v. Allow the gel to solidify and set it up in the tank filled with 1X TAE buffer.
vi. Load 2μl of DNA onto the gel and run at 100V for 30 minutes along with a positive control
1μl lambda DNA or any other high-molecular weight DNA.
vii. Observe the gel in the gel documentation system and record the results.
4. Results: Qualitative and quantitative DNA analysis would tell about the amount and purity of the
DNA.
5. Scope of the result: DNA isolation depends on the accuracy during mixing of various reagents.
DNA pellet must be handled carefully.
6. Results Required: High quality and good quantity of DNA
7. Cautions:
a. Care should be taken to do the operations as gently as possible. Vortexing, pipetting
using fine tips etc. should be avoided to prevent the shearing of DNA.
b. Phenolic chloroform extraction step may be repeated if the aqueous layer is not clear
and transparent
8. Suggested Readings: Experiments in Molecular Biology: Biochemical Applications by Zachary
F. Burton, Academic Press Inc; Spl edition (2003).
LMBTY614 7
