BASIC MOLECULAR BIOLOGY PRACTICAL
FOR THE STUDENTS OF
DEPARTMENT OF MEDICAL MICROBIOLOGY
NOBEL COLLEGE
POKHARA UNIVERSITY
Jul 1-6, 2007
Prepared by
Kiran Babu Tiwari
Asst. Prof. of Microbiology, USC
Research Scientist, RLABB
In association with
Upendra Thapa Shrestha
Nirajan Bhattarai
Vijayendra Agrawal
(Research Scientists, RLABB)
Reference:
Professor Dr. Vishwanath Prasad Agrawal
Executive Director
Research Laboratory for Biotechnology and Biochemistry (RLABB)
www.uscollege.edu.np/rlabb
http://www.rlabb.com.np/
http://www.vpagrawal.com/
Acknowledgement
We are indebted to
Prof. Dr. Vishwanath P. Agrawal,
Executive Director, Research Laboratory for Biotechnology and Biochemistry (RLABB);
Director, Universal Science College; and
Academician, National Academy of Science and Technology (NAST)
for providing the facilities to conduct the experiments enlisted in this manual.
CONTENTS
TITLE PAGE
ACKNOWLEDGEMENT
CONTENTS
JULY 1:
JULY 2:
EXPT 2. ISOLATION AND PURIFICATION OF RNA
JULY 3:
EXPT 5. RESTRICTION DIGESTION OF DNA
JULY 5:
JULY 6:
1. Principle of nucleic acids isolation from bacteria
Nucleic acids are present as nucleoprotein complexes in cells. The major problems encountered in isolation of pure and intact DNA or RNA molecules are: degradation of high molecular weight nucleic acids by mechanical damage or by hydrolytic action of nucleases, contamination of DNA preparations with RNA and vice versa and contamination of nucleic acids with proteins, polysaccharides and other high molecular weight compounds. Methods have been devised for isolation of nucleic acids from different sources taking adequate precautions to eliminate or minimize the above problems.
The main steps involved in isolation of nucleic acids are:
(a) Disruption of cells: Disintegration of bacterial cells can be achieved by treating them with cell coat hydrolyzing enzyme, i.e. lysozyme in presence of a detergent. at low temperature in buffers containing EDTA (chelates Mg2+ ions which are required for DNase activity). For isolation of RNA, and inhibitor of RNase such as bentonite, diethylpyrocarbonate, placental RNase inhibitor etc. is included in the extraction buffer.
(b) Dissociation of nucleo-protein complexes: The approach employed is such that the proteins either get dissociated or degraded while nucleic acids remain unaffected and intact. This is generally achieved by using detergents like SDS, phenol or broad-spectrum proteolytic enzymes such as pronase or proteinase K. Alkaline pH and high concentration of salts improve efficiency of the process.
(c) Removal of contaminating materials and precipitation of nucleic acids: Proteins are removed by treatment with phenol or mixture of chloroform-isoamyl alcohol or phenol-chloroform. Upon centrifugation, the denatured proteins form a layer at the interface between upper aqueous and lower organic phases, lipids and other contaminants remain in the same organic phase while nucleic acids are recovered in the aqueous phase from which they are precipitated with ethanol. For isolation of DNA, the contaminating RNA is removed by selective salt precipitation or treatment with DNase-free RNase. Conversely while isolating RNA, the preparation is incubated with DNase for eliminating DNA as an impurity.
(d) General precautions while handling nucleic acids:
1. All glasswares and solutions (except organic solvents) should be sterilized.
2. Gloves should be worn to avoid contamination of the experimental material and apparatus with nucleases with occur in fair abundance in skin exudates.
3. Phenol causes severe burns and phenol-containing solutions should, therefore, be handled with care. Thoroughly rinse the burns with large volume of water. Do not use ethanol.
4. For work with RNA, rinse all glasswares with 1% diethylpyrocarbonate solution to inactivate RNase and then autoclave them.
2. Basic components for Molecular Biology experiments
LB (Lauria-Bertani) broth: 0.5% NaCl, 1% Tryptone, 0.1% Yeast extract
Overnight Log- phase culture
Solution I (Lysis buffer): 25mM Tris, 50mM Glucose, Lysozyme [10mg/ml stock; GNB (Gram Negative Bacteria) 0.5mg/ml, GPB (Gram Positive Bacteria) 3-5mg/ml], pH 8.0
Solution II (Lysis): (a) for plasmid isolation: 0.2M NaOH, 10% SDS (b) for genomic DNA isolation: 10% SDS only.
Proteinase K: 10mg/ml stock, 10-20mg/65°C/3hrs
Solution III: 3M Sodium acetate, pH 4.8 by acetic acid
Phenol: distilled, protein precipitant
Chloroform: protein precipitant, phenol solvent
RNase: 5mg/ml stock, 1-2ml
Absolute ethanol/Isopropanol: DNA precipitant, 2.5vol of ethanol or 1vol of isopropanol
70% ethanol: washing DNA precipitant
TE buffer: DNA resuspending buffer, 10mM Tris, 1mM EDTA, pH 8.0; Autoclave before use
TAE buffer: Electrophoresis, 50X, Tris, 24.2gm; acetic acid, 5.71ml; EDTA (0.5M), 11.1ml; DW, 100ml; pH 8.0; Autoclave before use
Loading dye: 6X; 50% Glycerol, 6.0ml; 2%BPB (Bromophenol Blue), 1.0ml; DW, 3ml; Always use sterile DW
Ethidiun Bromide (EtBr)*: 10mg/ml stock (Final concentration: 0.5mg/ml)
λ/HindIII Marker: 23.13Kb, 9.42Kb, 6.56Kb, 2.32Kb, 2.07Kb, 0.56Kb and 0.13Kb
Restriction enzymes: EcoRI and HindIII (10U/ml each)
Restriction enzyme buffers for EcoRI and HindIII (10X each)
Sterile Double distilled water (DDW)
Water bath
Cold centrifuge (upto 20000rpm)
Micropipettes and sterile tips
*Note: EtBr is carcinogen, so, handle with gloves
Day 2
Expt. 1. Genomic DNA extraction from bacteria
-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT (Microfuge tube) and spin at 5000rpm for 10min.
-Remove the supernatant and spin once with same volume as above to collect more cell mass.
-Add 100µl Sol. I and keep for 30min at RT (Room temperature).
-Add 1/10 vol. of 10% SDS and swirl to mix.
-Add Proteinase K (1-2 µl) and incubate for 30min at 37ºC with gentle shaking.
-Add 1-2µl of RNase and incubate for 10min at RT.
-Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10min at RT.
-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.
-Add equal vol. of 3M sodium acetate, mix and stand it for an hour in cold.
-Spin at 10000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.
-Dissolve the pellet collected during spin in 50µl TE and store in deep freeze.
Expt. 2. RNA extraction from bacteria
-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000rpm for 10min.
-Remove the supernatant and spin once with same volume as above to collect more cell mass.
-Remove the supernatant.
-Add 100µl Sol. I and keep for 30min at RT.
-Add 1/10 vol. of 10% SDS and swirl to mix.
-Add Proteinase K (2 µl) and incubate for 30min at 37ºC with gentle shaking.
-Add equal vol. of Phenol:Chloroform (3:1) solution and mix gently.
-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.
-Add equal vol. of 3M sodium acetate and/or two vol. of absolute ethanol. Mix and stand it for 1-2 hour in cold.
-Spin at 15000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.
-Dissolve the pellet collected during spin in 50µl TE (pH 7.0) and store in deep freeze.
Day 3
Expt 3. Plasmid DNA extraction from bacteria
-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000rpm for 10min.
-Remove the supernatant and spin once with same volume as above to collect more cell mass.
-Remove the supernatant.
-Add 100µl Sol. I and keep for 30min at RT.
-Vortex for a while and add Proteinase K (2µl) and incubate for 30min at 37ºC with gentle shaking.
-Add freshly prepared Sol. IIa (200µl) and mix gently (Do not vortex).
-Add ice cold Sol. III (150µl) and mix gently (Do not vortex).
-Add 1-2µl of RNase and incubate for 10min at RT.
-Add equal vol. of Phenol: Chloroform (1:1), mix gently and keep for 10min at RT.
-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.
-Add equal vol. of isopropanol and stand it for an hour in cold.
-Spin at 13000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.
-Dissolve the pellet collected during spin in 50µl TE and store in deep freeze.
Day 4
1. Remove the DNA preparation from the freeze and thaw it.
2. Transfer 495μl TE buffer to a quartz cuvette and add 5μl of the DNA preparation. Mix well.
3. Set the spectrophotometer to 260nm and blank the instrument with TE buffer.
4. Measure the absorbance (A260) of the DNA dilution.
5. Repeat steps 3 & 4 with the spectrophotometer set at 280 nm.
6. Calculate the DNA concentration from A260. [µg/ml = A260 X dilution X 50].
7. Calculate A260/A280 in order to estimate the purity of the DNA preparation (Pure DNA has a A260/A280 ratio of 1.8 – 2.0).
1. Dilute the DNA extracts, Marker/s and enzymes in suitable solvents accordingly.
2. Calculate the volume for digestion reaction as shown in the table given below.
Components............ Rxn. .........1 Rxn. 2
λ DNA (1µg/µL) ............10 µL ..............-
DNA extract (1µg/µL) ...- ....................10 µL
HindIII (10U/µL) .........1 µL ................1 µL
HindIII buffer (10X) ....1 µL ................1 µL
DW ..................................8 µL ................8 µL
Total ...............................20 µL ..............20 µL
3. Perform digestion reaction by mixing the components in sterile MFT.
4. Briefly centrifuge the contents and keep at 37ºC for 6hrs.
5. Perform agarose gel electrophoresis to interpret the results.
Day 5
Expt. 6. Agarose electrophoresis of DNA
1. Prepare 0.8% agarose gel in 1X TAE buffer (28ml).
2. Dissolve agarose completely in micro-oven and cool to 600C.
3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5µg/ml.
4. Position a comb in the mold. Pour into gel mold and let it cool for 30 minutes.
5. Pour the TAE buffer into the gel buffer reservoir.
6. Prepare sample taking 20µl of DNA sample and mix with 4µl blue juice.
7. Carefully remove the comb.
8. Load the DNA (15 µl) in the wells, flanking wells with similarly processed DNA size standard.
Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.
9. Put the lid on the gel apparatus; attach the electrodes and adjust voltage to 100 volts.
10. Allow the gel to run until line of blue juice is visible near the end of the gel.
11. Turn off the current and visualize the gel in UV transilluminator.
12. Interpret the results.
Day 6
Expt 7. RAPD-PC(Randomly Amplified Polymorphic DNA – Polymerase Chain Reaction)
1. Thaw the DNA extract and dilute in sterile TE to final concentration with10ng/µl.
2. Prepare reaction mixture as given below:
Components---------- Volume (µl)
PCR buffer (pH 8.3) ----------5
dNTP (2.5mM each) ---------4
Taq polymerase (1U/µl) -----1
Template DNA (10ng/µl) ----1
RAPD-Primer (10µM) -------8
10% DMSO ------------------5
DDW ------------------------26
Final Volume-----------------50
3. Operate the thermal cycler program as given below:
Initial denaturation------- 94ºC-------5mi -----Single step
Denaturation -------------94ºC -------1min
Annealing ----------------36ºC -------1min ----30 Cycles
Extension ----------------72ºC -------2min
Final extension -----------72ºC -------5min ----Single step
4. Perform agarose gel electrophoresis as described in Expt. 6.
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