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10. PLASMID ISOLATION
Bacterial plasmids are double stranded closed circular DNA molecules that
range in size from 1Kb to more than 1000Kb. They are found in variety of
bacterial species, where they behave as accessory genetic units that
replicate and inherited independent of the bacterial chromosome. Natural
E. coli isolates often carry plasmids specifying resistance to
antibiotics, resistance to heavy metals, sensitivity to mutagens, production
of rare amino acids etc.,
Many plasmids were constructed in the laboratory with fragments of DNA from
the naturally occuring plasmids. These artificial plasmids and their
derivatives are the most commonly used vectors in recombinant DNA work. All
plasmids used as cloning vectors contain three common features; an origin of
replication, a selectable marker gene and a cloning site.
Alkaline lysis procedure is the most commonly used protocol for plasmid
isolation. Bacteria are lysed by treatment with a solution containing sodium
dodecyl sulfate and NaoH (SDS denatures the bacterial proteins, NaoH
denatures chromosomal and plasmid DNA). The mixture is neutralized with
potassium acetate, causing the covalently closed plasmid DNA to reanneal
rapidly. Most of the chromosomal DNA and bacterial proteins precipitate as
does SDS, which forms a complex with potassium and are removed by
centrifugation. The remaining proteins are removed by phenol:chloroform
extraction. Then the plasmids are precipitated with 100% ethanol.
MEDIA AND SOLUTIONS
LB medium
Tryptone :10gm
Yeast Extract :5gm
NaCl :10gm
Dissolve in 750ml of distilled water, adjust the pH to 7.0 with 4N NaOH and
make up the volume to 1000ml with distilled water .
STOCK SOLUTIONS
1M Tris
:Dissolve 121.1gm of tris base in 800ml of water .Adjust the pH to 8.0 with
HCl ,adjust final volume to1000ml.
EDTA(0.5M):
Add 186.1gm of EDTA salt to 750ml of sterile water ,stir vigorously on
magnetic stirrer .Adjust the pH to 8.0 with NaOH .Make volume up to 1000ml.
10%SDS
:Dissolve 10gm of SDS in 90ml distilled water .Heat to 68oC to
dissolve .Adjust the pH to 7.2 by adding a few drops of HCl .make the volume
to 100ml with water .
4N NaOH:
Dissolve 16gm of NaOH pellets in 50ml water and make up the volume to 100ml
with water .
3M Sodium acetate
:Dissolve 408.1gm of sodium acetate .3H2O in 800ml of
water.Adjust the pH to 5.2 with glacial acetic acid .Adjust the volume to 1L
.Sterilize by autoclaving .
RNase:Dissolve
RNase A at a concentration of 10mg/ml in 10mM Tris (pH7.5),15mM NaCl .Heat
to 1000C for 15 minutes.Allow to cool slowly to room
temparature.Store at –200C.
Solution – I
50 mM
Glucose
25 mM
Tris. Cl (pH 8.0)
10 mM EDTA
(pH 8.0)
Dissolve .9gm of glucose in 80ml of distilled water to this add 2.5ml of 1M
Tris (pH8.0)and 2ml of .5ml EDTA (ph 8.0).Make up the volume to 100ml with
distilled water .
Solution – II
0.2 N NaoH
1 % SDS
Solution – III
5M
Potassium acetate 60 Ml
Glacial
aceticacid 11.5 Ml
Water 28.5
Ml
Take 60ml of 5M potassium acetate ,add 11.5ml of glacial acetic acid and
28.5ml of water .The resulting solution is 3m with respect to potassium ,5m
with respect to acetate .
Absolute ethanol
70% ethanol
10mg/ml DNase free RNase
T10E0.1
Phenol (pH 7.0) Tris saturated
Chloroform: Isoamylalcohol (24:1)
3M Sodium acetate.
Protocol
1)
Transfer a single bacterial colony in to a 2ml of LB medium containing the
70mg/ml Ampicillin. Incubate the culture overnight at 370C with
vigorous shaking.
2)
Pour 1.5 ml of the culture into a microfuge tube.
Centrifuge at 10,000 rpm for 5 minutes.
3)
When centrifugation is complete, decant the medium leaving the bacterial
pellet as dry as possible.
4)
Resuspend the bacterial pellet in 100
μl
of ice-cold solution 1 by vigorous vortexing. Keep on ice.
5)
Add 200
μl
of freshly prepared solution II to each bacterial suspension. Close the
tube tightly and mix the contents by inverting the tube rapidly five times.
Store the tube on ice.
6)
Add 150
μl
of ice-cold solution III. Close the tube and disperse Solution III through
the viscous bacterial lysate by inverting the tube several times. Store the
tube on ice for 3-5 minutes.
7)
Centrifuge the bacterial lysate at 10000rpm for 5 minutes at 40C
in microfuge. Transfer the supernatant to a fresh tube.
8)
To the supernatant add 5
μl
of 10 mg/ml DNase free RNase, incubate at 370C for 1 hour.
9)
Add an equal volume of phenol:chloroform : iso amylalcohol(25:24:1). Mix the
organic and aqueous phases by vortexing and then centrifuge the emulsion at
10,000 rpm for 5 minutes. Transfer the aqueous upper layer to a fresh tube.
10)
Add equal volume of chloroform : isoamylalcohol (24:1)to the aqueous phase
and mix it by inverting. Centrifuge the emulsion at 10,000 rpm for 5
minutes. Transfer the aqueous upper layer to a fresh tube.
11)
Add 1/10th volume of 3M Sodium acetate and 2.5 volumes of
ethanol to the aqueous layer, Mix vigorously. Keep the tube in –700C
for 1 hour.
12)
Collect precipitate DNA by centrifugation at 12,000 rpm for 12 minutes.
Decant the ethanol .
13)
Wash the pellet with 1ml of 70% ethanol by centrifuge at 12,000rpm for 5
minutes .
14)
Decant the 70% ethanol and dry the pellet at 650C till the
alcohol evaporates completely.
15)
Dissolve the DNA pellet in 50
μl
of T10E0.1. Store the DNA at –200C
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