DNA Extraction: Introduction, Test Requirements, Test Procedure and

DNA Extraction: Introduction, Test Requirements, Test Procedure and Observation

Introduction of DNA Extraction

The availability of genomic DNA (gDNA) from microorganisms and its DNA extraction is necessary for cloning genes and selecting recombinant constructs and for taxonomy and diagnostics. This method for DNA extraction from bacteria ( Gram-positive or Gram-negative bacteria ) or yeasts is as follows. This method includes using SDS/CTAB/proteinase K for lysis of the cells and the supernatant is extracted with chloroform to remove traces of isopropanol again centrifuge. Washing with 70% ethanol is mandatory to remove salt from the DNA. Centrifuge and discard the ethanol, drying on the benchtop at room temperature. Finally, resuspend the pellets in TE buffer and keep it at 4°C. But here we concern with bacterial DNA extraction

Requirements for the DNA Extraction

The followings are the requirements for this test procedure-

Test spacemen (bacteria culture)
TE buffer
Vortex mixer
SDS solution
Proteinase K
5M NaCl
10% CTAB
0.7 M NaCl
Isoamyl alcohol
70% Ethanol
Chloroform
1.5mL Eppendorf tubes
Centrifuge
Chloroform
Micropipette and tips

The Procedure of DNA Extraction

For liquid culture, spin down 1-3 ml of culture and remove the media. Add 567 µl of TE buffer to the pelleted cells. If agar media are used, choose one colony from the petri dish ( using a sterile toothpick) and place the toothpick with the colony into a sterile Eppendorf tube containing 567µl of TE buffer. Re- suspend the pellet by repeated pipetting or by gentle vortexing the toothpick with the solution so that the cells become resuspended. Add 30 µl of 10% SDS and 3µl of a 20 mg/mL solution of proteinase K. Mix and incubate for 1 hour at 37°C. After incubation, add 100µl of 5 M sodium chloride and mix. Afterwards add 80µl of a CTAB ( 10%)/NaCl (0.7M) solution. Incubate this solution at 65°C for 10 minutes. After incubation, add an equal volume of chloroform: isoamyl alcohol (24:1) and again mix. Centrifuge for 5 minutes and transfer the aqueous solution to a new tube. Be careful not to transfer the interface. Add another equal volume of chloroform and isoamyl alcohol and mix well. Centrifuge at 14000 rpm for 5 minutes and transfer supernatant to a new tube. Repeat the first extraction again (chloroform: isoamyl alcohol alone). Add 0.6 ml of isopropanol and mix gently until the DNA precipitates. Centrifuge and remove isopropanol. Add 1 mL of 70% ethanol to wash the salt away from the DNA. Centrifuge and discard the ethanol, drying on the benchtop at room temperature. Resuspend the pellet in 50-100µl of TE buffer and keeps it at 4°C.

Observation of a Step During DNA Extraction

DNA precipitation is a step of DNA isolation. Just above the bottom in a tube and middle part of fluid a white-like shining substance demonstrating DNA precipitate.

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