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BT - Practical 5 & 6

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21 views6 pages

BT - Practical 5 & 6

Uploaded by

Gounder Kirthika
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© © All Rights Reserved
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Practical 5: Plasmid DNA isolation from E.

coliusing alkaline lysis method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic


cells. In addition to chromosomal DNA, bacterial cells contain a small circular closed double
stranded DNA molecule having size ranging from 1kb to 200kb. Mostly plasmids contain
genes that are used to tide-over extreme conditions like antibiotic resistance, DNA repair
mechanism system etc. Plasmids constructed in the laboratory are mainly used as cloning
vectors. These synthetic plasmids contain only essential features such as ori site, antibiotic
resistance marker, multiple cloning site, etc. There are so many protocols available for
plasmid isolation. In this experiment, we will use alkaline lysis method of Birnboim and Doly
in 1979.

Principle

Exposure of the bacterial suspension to a strongly anionic detergent at high pH


disrupts the cell wall, denatures the chromosomal DNA and proteins and releases the plasmid
into supernatant. Although the alkaline solution completely disrupts base pairing, the strands
of closed circular plasmid DNA are unable to separate from each other because they are
topologically intertwined. As low concentration and short duration of exposure of NaOH is
given, plasmid DNA is renatured when the appropriate conditions are given. During lysis,
bacterial proteins, broken cell wall and denatured chromosomal DNA become enmeshed in
large complexes that are coated with SDS. These complexes are efficiently precipitated from
solution when sodium ions are replaced by potassium ions. After the denatured material has
been removed by centrifugation, native plasmid DNA can be recovered from the supernatant.
Two strands of covalently closed circular plasmid DNA are unable to separate when the lysis
solution is given because of intertwining complexity. It also renatures immediately and
completely when conditions are returned to normal while the genomic DNA forms an
aggregate with SDS and proteins.
Separation on the basis of conformation by Alkaline denaturation method:

The basis of this technique is that there is a narrow pH range at which non-
supercoiled DNA is denatured, whereas super coiled plasmids are not. If sodium hydroxide is
added to a cell extract, so that the pH is adjusted to 12.0-12.5, then the H-bonding in non-
super coiled DNA molecules is broken, causing the double helix to unwind and the two
polynucleotide chains to separate. If acid is now added, these denatured bacterial DNA
strands re-aggregate into a tangled mass. Centrifugation will pellet this insoluble mass
leaving plasmid in the supernatant.

Reagents:

1. SOLUTION 1
a) 25mM Tris-Cl (pH 8 )
b) 10 mM EDTA (pH 8)
c) 50uM Glucose

2. SOLUTION 2 (Alkaline lysis solution1) prepare immediately before use.


a. 0.2 N NaOH
b.1% SDS
3. SOLUTION 3
a. 7.5 M ammonium acetate (pH 5.2) [note : pH is adjusted with glacial acetic acid ]
4. TE Buffer.
a. 10 mMTris-Cl (pH 8) (121.14 g tris Base in 1L d/w, adjust pH 8 with conc. HCL
b. 1mM EDTA (pH 8) (dilute from 5mM EDTA stock)
5. LB agar medium and LB broth
6. Ampicillin (100μg/ml)
7. 95% ethanol and 70% ethanol
8. Loading dye: 40% glycerol + 0.5% Bromo phenol blue
Procedure:

DAY 1: Inoculate loopful of culture from glycerol stock in LB broth containing Ampicillin
(100μg/ml) and incubate at 37oC for 24 hrs.
DAY 2: Refer to the following steps carefully.
Procedure
1. Take 2 ml of culture in Eppendorf tube.
2. Centrifuge at 6000rpm for 12 min at 4oC.
3. Decant the supernatant.
4. Resuspend the pellet in 100μl of Ice Cold Solution I. Mix gently. Incubate for 5min at
RT
5. Add 200μl of freshly prepared Solution II.
6. Mix the contents by gentle inversion.
7. Keep the eppendorf tubes on ice for 5 min.
8. Add 500μl of ice cold Solution III.
9. Mix by inverting the tubes and store on ice 5 min.
10. Centrifuge at 5000rpm for 10 min. Take the supernatant in another tube.
11. Add 50μl of Solution III and 300μl of 70% Ethanol in this supernatant and mix gently.
Keep on ice for 15 minutes (DNA appears as white precipitate).
12. Centrifuge at 5000rpm for 3 minutes.
13. Carefully drain off the supernatant.
14. Dry the pellet. When the pellet turns transparent, add 30μl of TE buffer to re-suspend and
store at 4oC.
15. Sample preparation for gel loading: Take 8ul of sample and 2ul of loading dye mix gently
and load 10ul in wells.
Practical 6: To resolve the purified samples of plasmid and genomic DNA using agarose
gel electrophoresis.

Introduction
Agarose gel electrophoresis is a widely used method that separates molecules based
on charge, size and shape. It is particularly useful in separating charged molecules such as
DNA and RNA. AGE poses high resolving power yet is relatively simple and straight
forward to perform. The applications of this simple technique are many viz.
(1) Gene cloning and analysis of DNA
(2) Restriction mapping of DNA molecules
(3) Molecular weight determination of DNA
(4) Analysis of PCR products

Principle
When agarose is melted and then cooled in an aqueous solution, it forms gel by
hydrogen bonding. The population of DNA fragments in given sample will move through an
agarose gel under the influence of an electric field, where negatively charged DNA molecules
will be drawn to the anodes. Their rate of movement is based almost entirely on size, with the
largest molecules having the lowest mobility. The concentration of agarose in the gel
determines pore size, and a DNA fragment having a particular size will migrate at different
rates through gels of different concentrations. There is a linear relationship between the log of
mobility and gel concentration over a certain range of fragment sizes, so a gel concentration
must be chosen that will effectively separate the molecules in the DNA population. Gels of
0.7% (w/v) agarose are suitable for separating linear DNA molecules 0.5-10 kb in size. The
log molecular weights of the known marker fragments, such as lambda phage DNA cut with
the HindIII, can be plotted against mobility. The resulting bands can be used to determine the
molecular weights of unknown DNA fragments. As a quick measure, the molecular weight of
an unknown fragment can be estimated by direct comparison the position of lambda
fragments on the gel. The bands are visualized by ultraviolet (UV) light illumination after
staining with the fluorescent dye, Ethidium bromide.
Materials:

1. 50X TAE – 242g Tris base

57.1 ml of glacial acetic acid

100ml of 0.5mM EDTA

Make up volume 1L

pH - 8.0

Working solution: Dilute stock solution 50 times to get 1X concentration

2. 1.2% Agarose in 1X TAE (dissolve 1.2 gmagarose in1X TAE)

3. Tracking dye – 40% glycerol+ 0.5% Bromo-phenol blue

4. 10mg/mlEtBr –dissolve 10mg EtBr in 10-15ml of 95% alcohol. Then make up


volume

5. Electrophoresis unit with power supply

Procedure:

1. Prepare 1.2% Agarose in 1X TAE. Use microwave oven for proper dissolving
2. Allow to cool it then add 2ul/100ml of EtBr from 10mg/ml stock. Mix it properly.
3. Set up electrophoresis unit.
4. Pour gel in gel caster with comb. (avoid bubbles in gel)
5. Allow to solidify.
6. Remove comb dentally.
7. Keep gel cast in reservoir tank having 1X TAE buffer.
8. Load prepared sample. ( 8ul sample + 2ul of tracking dye)
9. Switch on unit. (run on 50V)
10. Allow ¾ run of tracking dye then stop run.
11. Take gel out of unit and observe under UV

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