Bacterial DNA Isolation:

Aim

To isolate genomic DNA from Bacterial Cell.

Steps Involved:
1) Re suspension and cell wall lysis;
2) Purification by phase separation;
3) Precipitation
4) Washing;
5) Storage;
6) Agarose gel electrophoresis;

Chemicals Required:
1) TE Buffer: Tris EDTA Buffer
10mM Tris HCl and 1mM EDTA.
2) 10% sodium Dodecyl Sulphate
3) Phenol: Chloroform 50:50
4) Isopropanol
5) 3M sodium Acetate
6) Proteinase K.

Principle

DNA is intra cellular in nature and to isolate it firstly:

1) Cell Wall lysis is to be done and for this hyper suspension detergents
like SDS (Sodium Dodecyl Sulphate) and enzimes (Proteinase K) are
used which dissolves proteins, lipids and phospholipids components.

2) Protein removal and phase separation: For this phenol and

chloroform is used in equal amounts in which phenol is a protein
separator and chloroform acts as a phase separator.

3) DNA Precipitation: For this potassium acetate and ice cold iso-
propenal are used because DNA is insoluble and precipitates out.

Procedure:

1) Re-suspension and Cell Wall lysis

 1.1) Take overnight grown bacterial culture 1- 1.5 ml in eppendorf
tube.
1.2) Spin the culture at 5,000 rpm for 5 mins;
1.3) Discard the supernatant and to the pallet add:

1.3.1) 467 ul TE Buffer (Re-suspension Buffer)

1.3.2) 30 ul 10% SDS;
1.3.3) 3 ul Proteinase K.
Incubate the mixture for 1 hr at 370 C.

2) Purification By Phase Separation:

2.1) Add equal volume of phenol : choloroform i.e. 500 ul in 1:1 ratio.
2.2) Mix gently the whole mixture by inversion for 10 mins;
2.3) Centifuge the mixture in the eppendorf at 10,000 rpm for 10 mins.
2.4) Transfer the top layer to the new tube (genomic DNA )

3) Precipitation:
3.1) Add 1/10th volume of 3M potassium acetate (Ph 5.2- 5.5) and equal
amount of ice cold iso-propanol.
3.2) Incubate at 00C for 30 mins may be overnight.

4) Washing:
4.1) Centrifuge the incubated mixture at 10,000 rpm for 10 mins.
4.2) Discard the supernatant and to the palette add 200 ul of 70%
ethanol.
4.3) Centrifuge it at 10,000 rpm for 5 mins.
4.4) Discard the supernatant and allow the palette to dry to remove the
smell of ethanol.

5) Storage
Dissolve it in appropriate amount of TE Buffer and go for agarose gel
electrophoresis.
Observation:

Pink colored DNA bands were observed under U.V. Transilluminator.

CTAB Method for genomic DNA Isolation from Plant
Cells

Aim:

To Isolate Genomic DNA from Plant Cells by CTAB Method

Re-agents required:
1) CTAB buffer- 5 ml: CTAB 100 µl (2% CTAB, 20 mM EDTA, 100 mM Tris
HCl and 1.4 M Nacl)
2) β Mercaptoethanol 10 µl.
3) Choloroform: Isoamyl alcohol (24:1)
4) Chilled Isopropanol
5) 70% Ethenol
6) TE Buffer 20 µl.

Procedure:
1) Grind 1 gm of washed and dried plant samples in 5 ml pre heated
CTAB Buffer and add 0.2 % β Mercaptoethanol (10 µl)
2) Transfer 500 µl homogenate into eppendorf tube.
3) Incubate it at 600C for 1 hr.
4) Mix gently by inversion after 15 mins interval during the incubation
time.
5) After this add equal amount of chloroform: Isoamyl alcohol (24:1)
6) Mix by inversion by 15 mins
7) Spin at 10,000 rpm for 10 mins
8) Transfer the supernatant in a fresh eppendorf and add equal volume of
chilled isopropanol and incubate over night at 40C.
 9) Spin at 10,000 rpm at 10 mins.
10) Discard the supernatant and wash the pallet with 70%
ethanol( Add 300 µl of 70% ethanol and spin at 5,000 rpm for 10 mins)
11) Discard the supernatant and air dry the pallet.
12) Add 20 µl of TE Buffer and go for agarose gel electrophoresis.

Observation:
Pink colored DNA bands were observed under UV Transilluminator.
 Plasmid Isolation
AIM:

To isolate plasmid from bacterial cell.

Reagents and Buffers Required:

1) P1 Buffer: Re-suspension Buffer
50 mM Tris HCl
10 mM EDTA
100 μg per ml RNAase
2) P2 Buffer: Lysis Buffer
200 mM NaOH
1% SDS
3) P3 Buffer: Neutralization Buffer
3M Potassium Acetate
Phenol: Chloroform: Isomamyl alcohol (25:24:1)
Chloroform: Isomamyl alcohol (24:1)
Isopropanol
70% Ethenol

Principle:
Plasmid is extra chromosomal, circular and self replicating material and is
intracellular and hence to isolate is cell wall lysis is to be done which is done
by using detergents(SDS) which dissolves lipids, phospholipids and proteins
present in the cell wall as well as NaOH is used to denature the bacterial
genomic DNA.

Procedure:
1) Take overnight grown bacterial culture 1- 1.5 ml in a eppandorf.
2) Centrifuge it at 5,000 rpm for 5 mins.
3) Discard the supernatant and to the pallete and 100 μl P1 Buffer and 5
μl RNAase.
4) Incubate it at 00c for 5-10 mins.
5) Then add 200 ml of P2 Buffer and incubate it at 00C for 5-10 mins.
6) Then add 150 μl P3 Buffer and again incubate it at 00C for 5 mins.
7) Centrifuge the mixture at 10,000 rpm for 10 mins
 8) Transfer the supernatant in which plasmid is present in a new
eppendorf and add equal volume of phenol: chloroform: isoamyl
alcohol in ratio 25:24:1.
9) Mix it for 10 mins and then centrifuge at 10,000 rpm for 10 mins.
10) Transfer top layer and to it add equal volume of chloroform:
Isoamyl alcohol in ratio 24:1
11) Mix for 10 mins and spin it at 10,000 rpm for 10 mins.
12) Transfer top layer and add 1/10th volume of potassium acetate
(3M, Ph 5.2-5.5) add equal volume of ice cold isopropanol.
13) Incubate it at 00C for overnight.
14) Then add 70% ethanol 200 μl.
15) Store in TE Buffer.

Observation:
Pink colored bands were observed in UV Trans-illuminator.