Exp. No.7 Extraction and separation of chromosomal DNA from Bacteria

  Extraction and separation of chromosomal DNA from Bacteria

Introduction

            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 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 in order 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. 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 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.

Aim

To isolate the genomic DNA from E. coli cells

Materials and Methods

LB Broth, E. coli cells, Reagents, TE buffer (pH 8.0), 10% SDS, Proteinase K, Phenol-chloroform mixture, 5M Sodium Acetate (pH 5.2), Isopropanol, 70% ethanol, Autoclaved Distilled Water, Eppendorf tubes 2 ml, Micropipette, Microtips, Microfuge

Preparation of Reagents:

1. TE Buffer (pH 8.0): 10 mm TrisHCl (pH 8.0), 1 mm EDTA (pH 8.0)

·         For a 1 M solution, dissolve 12.1 g of Tris base in 80 mL of nuclease-free water.

·         Adjust the pH to 8 values by slowly adding approximately 6-7 mL concentrated HCl. Adding concentrated HCl to the Tris buffer will increase the temperature of the solution, which affects the pH. Allow the solution to cool to room temperature before making final adjustments to the pH (using more HCl if necessary).

·         Adjust the volume of the solution to 100 mL with water

·         To obtain a 10 mM Tris-HCl pH 8 solution, dilute 1 M Tris-HCl 1:100 with nuclease-free water. For example, add 1 mL of 1 M Tris-HCl to 99 mL of nuclease-free water.

Reagent	Volume	Final concentration
1M Tris-Cl (pH 8.0)	1 mL	10 mM
0.5M EDTA (pH 8.0)	0.2 mL	1 mM
Distilled H2O	98.8 mL

 

2. 10% SDS: Dissolve 10 g of SDS in 100 ml autoclaved distilled water.

3. Proteinase K: Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled water.

4. Phenol – Chloroform Mixture: The pH is very important. For RNA purification, the pH is kept around pH 4, which retains RNA in the aqueous phase preferentially. For DNA purification, the pH is usually 7 to 8, at which point all nucleic acids are found in the aqueous phase. Mix equal volume of phenol with chloroform. Keep the mixture on ice and add 20 ml TE buffer, extract by shaking for 15 minutes. Remove the dust on the surface layer using a pipette. Repeat 4-5 times. Add 30-40 ml of TE buffer and store it on ice.

5. 5M Sodium Acetate: Dissolve 41 g of sodium acetate in 100 ml distilled water and adjust pH with dilute acetic acid (pH 5.2).

6. Isopropanol

7. 70% Ethanol

 Procedure

·         2 ml overnight culture was taken and the cells were harvested by centrifugation for 10 minutes

·         875 µl of TE buffer was added to the cell pellet and the cells were resuspended in the buffer by gentle mixing.

·         100 µl of 10% SDS and 5 µl of Proteinase K were added to the cells.

·         The above mixture was mixed well and incubated at 37º C for an hour in an incubator.

·         1 ml of phenol-chloroform mixture was added to the contents, mixed well by inverting and incubated at room temperature for 5 minutes.

·         The contents were centrifuged at 10,000 rpm for 10 minutes at 4º C.

·         The highly viscous jelly like supernatant is collected using cut tips and is transferred to a fresh tube.

·         The process was repeated once again with phenol-chloroform mixture and the supernatant was collected in a fresh tube.

·         100 µl of 5M sodium acetate was added to the contents and was mixed gently.

·         2 ml of isopropanol was added and mixed gently by inversion till white strands of DNA precipitates out.

·         The contents were centrifuged at 5,000 rpm for 10 minutes.

·         The supernatant was removed and 1ml 70% ethanol was added.

·         The above contents were centrifuged at 5,000 rpm for 10 minutes.

·         After air drying for 5 minutes 200 µl of TE buffer or distilled water was added

·         10 µl of DNA sample was taken and was diluted to 1 or 2 ml with distilled water.

·         The concentration of DNA was determined using a spectrophotometer at 260/280 nm.

·         The remaining samples were stored for further experiments.

Precautions:

·         Cut tips should be used so that the DNA is not subjected to mechanical disruption.

·         Depending on the source of DNA the incubation period of Proteinase K should extended.

·         The phenol chloroform extraction should be repeated depending on the source of DNA to obtain pure DNA.

·         DNase free plastic wares and reagents should be used.

Result

The sample should be observed in a UV visible spectrometer for its absorbance maxima. If the sample obtains a peak at the range of 260 nm then it is confirmed that the sample contains DNA in it.