DNA ISOLATION FROM BACTERIA

To Isolate Genomic DNA from Bacterial Cell

Theory:

  • In molecular biology the isolation and purification of DNA from cell is most important process.
  • The bacterial cell that is used in this procedure should be grown in suitable media under favourable conditions and harvested in late log to early stationary phase.
  • Along with DNA bacterial cell contain RNA, lipids, protein which needs to separate.
  • The first step in DNA isolation is to disrupt the cell membrane and this is done by SDS (Sodium Dodecyl Sulphate)
  • Endogenous nucleases present on human fingertips can degrade the nucleic acid during purification. This degradation by nucleases can be prevented by chelating mg2+ ions using EDTA.
  • Mg2+ ion is a necessary cofactor for the action of the nucleases.
  • Proteinase K is used to degrade protein in the disrupted cell.
  • And the function of phenol and chloroform is to denature and separate the protein from DNA.
  • The denatured protein makes a layer between aqueous and the organic phase.
  • DNA from the disrupted cell is precipitated by cold absolute ethanol or isopropanol.

Requirements:

  • LB media
  • E.coli DH5α cells
  • TE buffer
  • 10% SDS
  • Proteinase K
  • Phenol:Chloroform (1:1)
  • 5M Sodium acetate
  • Isopropanol
  • 70%ethanol
  • Autoclaved distilled water
  • Eppendorf tube
  • Micropipette
  • Microtips
Micro-centrifuge

Procedure:

  • Take 1.5ml of the bacterial culture (grown overnight) and harvest it by using centrifuge at 5000 rpm for 3-4 minutes.
  • Discard the supernatant and add 600μl lysis buffer and incubate at room temperature for 10 minutes.
  • Then add 200μl 10% SDS and 5μl proteinase K to the cells and incubate for 10-15 at 37o C.
  • Now incubate the tubes in water bath for 10 minutes at 60o C and immediately transfer to ice bucket and keep for 5 minutes.
  • Adding 500μl phenol: chloroform solution in 1:1 ratio and mix by inversion for 1-2 minutes, incubate for 5 minutes.
  • After incubation centrifuge the mix at 10000 rpm for 10 minutes at 4o C.
  • Now carefully transfer the upper aqueous layer to the fresh tube.
  • Add 100μl 5M sodium acetate and equal amount of isopropanol mix by inversion and store at-20o C for 1 hour or overnight.
  • Centrifuge the mix at 5000 rpm for 10 minutes and discard the supernatant.
  • Then add 1 ml of 70% cold ethanol and mix by inversion.
  • Again centrifuge at 5000 rpm for 10 minutes and discard supernatant.
  • Air dry the pellet and add 40μl TE buffer and store at 4o C for further use.

Precaution:

  • To avoid mechanical disruption of DNA cut tips should be used.
  • The incubation period of proteinase K depends on the source of DNA and should be extended.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: