ISOLATION AND SEPARATION OF CHROMOSOMAL DNA FROM BACTERIA

 ISOLATION AND SEPARATION OF CHROMOSOMAL DNA FROM BACTERIA

 AIM:

To isolate and separate the chromosomal DNA from bacteria

 

PRINCIPLE:

 

Genomic (chromosomal) DNA from either bacteria, plant or animal cell can be isolated by lysis method. For plant cell or bacterial cell, first cell wall is mechanically disrupted. The cell wall is broken enzymatically using enzymes like lysozyme. When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. After the addition of acetate- containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.DNA extraction involves lysing the cells and solubilizing DNA, which is followed by chemical or enzymatic methods to remove macromolecules, lipids, RNA, or proteins.

 

MATERIALS REQUIRED:

 

        Bacterial isolate cultured on an agar plate

        Inoculation loop

        Incubator

        Eppendorf tubes (vol 1 5 ml) sterilised

        Pipettes and sterilised tips

        Centrifuge

SOLUTIONS

 

        Chloroform: iso-amyl alcohol (24:1)

        Phenol/chloroform: iso-amyl alcohol (1:1 v/v)

        CTAB/NaCl: Mix 10% Hexadecyltrimethylammonium Bromide and 5M NaCl 5M (1:1).

Store at room temperature.

    RNAse (10 mg/ ml):Disolve 100 mg RNAse in 10 ml sterile distilled water and incubate at 90 ºC for 10 minutes to inactivate DNAss. Keep at – 20 ºC until use.

        Proteinase K (20 mg/ ml): Disolve 100 mg proteinase K in 5 ml sterile distilled water.

Keep at – 20 ºC.

        TE (pH 8.0): 10 mM Tris-HCl, 1mM EDTA. Autoclave and keep at room temperature.

    10% SDS (w/v): Disolve 10 g of SDS in 100 ml of distilled water. Keep at room temperature.

        5M NaCl: Disolve 292.2 g of NaCl in a final volume of 1 litre. Autoclave.

        Molecular biology grade water autoclaved

PROCEDURE

 

1.   Resuspend a loop of colonies from a cultured agar plate in 500 µl of TE and dissolve completely.

2.    Add 30 µl of 10% SDS. Mix well.

      3.   Add 3 µl of proteinase K (20 mg/ml). Incubate at 37 ºC for 60 minutes.

      4. Add first 100 µl of 5M NaCl mix well and then 80 µl of CTAB/NaCl. Mix by inverting the tube (gentle tilting) and incubate at 65ºC for 10 min.

                   5. To the solution add an equal volumes of phenol/chloroform:iso-amyl alcohol (25/24:1)

mix to emulsify and spin for 5 minutes at 8000 rpm. Pipette upper layer into a fresh tube..

                  6. Repeat step 5

                   7. Add RNAse to a final concentration of 50 µg/ml and incubate for 30 minutes at 37 ºC

       8. Repeat step 5

       9. Precipitate DNA adding 1/10 vols of 5M NaCl and 2 vols of 100% cold ethanol and keep at -20º C o/n or at -80º C for 60 minutes.

 

      10. Spin at 12.000 rpm for 20 minutes.

      11. Remove supernatant.

      12. Wash by adding 70% ethanol followed by centrifugation as in step 10. Dry completely.

 

             13. Redissolve in 50-200 µl of TE and keep at -20 ºC